Defensin-antigen fusion proteins

ABSTRACT

The present invention relates to a vaccine for increasing the immunogenicity of a tumor antigen thus allowing treatment of cancer, as well as a vaccine that increases the immunogenicity of a viral antigen, thus allowing treatment of viral infection, including immunodeficiency virus (HIV) infection. In particular, the present invention provides a fusion protein comprising a defensin fused to either a tumor antigen or viral antigen which is administered as either a protein or nucleic acid vaccine to elicit an immune response effective in treating cancer or effective in treating or preventing viral infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/233,074, filed Sep. 15, 2000, which application is incorporatedherein fully by this reference.

FIELD OF THE INVENTION

The present invention relates to a vaccine for increasing theimmunogenicity of a tumor antigen thus allowing treatment of cancer, aswell as a vaccine that increases the immunogenicity of a viral antigen,thus allowing treatment of viral infection, including immunodeficiencyvirus (HIV) infection. In particular, the present invention provides afusion protein comprising a defensin fused to either a tumor antigen orviral antigen which is administered as either a protein or nucleic acidvaccine to elicit an immune response effective in treating cancer oreffective in treating or preventing viral infection.

BACKGROUND OF THE INVENTION

Tumor cells are known to express tumor-specific antigens on the cellsurface. These antigens are believed to be poorly immunogenic, largelybecause they represent gene products of oncogenes or other cellulargenes which are normally present in the host and are therefore notclearly recognized as nonself. Although numerous investigators havetried to target immune responses against epitopes from various tumorspecific antigens, none have been successful in eliciting adequate tumorimmunity in vivo (71).

Humans are particularly vulnerable to cancer as a result of anineffective immunogenic response (72). In fact, the poor immunogenicityof relevant cancer antigens has proven to be the single greatestobstacle to successful immunotherapy with tumor vaccines (73). Over thepast 30 years, literally thousands of patients have been administeredtumor cell antigens as vaccine preparations, but the results of thesetrials have demonstrated that tumor cell immunization has failed toprovide a rational basis for the design or construction of effectivevaccines. Even where patients express tumor-specific antibodies orcytotoxic T-cells, this immune response does not correlate with asuppression of the associated disease. This failure of the immune systemto protect the host may be due to expression of tumor antigens that arepoorly immunogenic or to heterologous expression of specific antigens byvarious tumor cells. The appropriate presentation of tumor antigens inorder to elicit an immune response effective in inhibiting tumor growthremains a central issue in the development of an effective cancervaccine.

Anti-microbial peptides such as defensins have been identified as keyelements in the innate immunity against infection. Originally identifiedon the basis of their anti-microbial activity, defensins are expressedwithin tissues and cells that frequently encounter microorganisms, andare divided into the alpha- and beta-defensin subfamilies, distinguishedby cysteine residue pairing. Defensins have been suggested to play arole also in inflammation, wound repair, and regulation of the specificimmune response. They induce expression of cytokines and chemokines,production of histamine and modulation of antibody responses, and theyhave been found to be associated with HLA-DR molecules and withlipoprotein (a) (130).

There remains a great need for a method of presenting tumor antigens,which are known to be poorly immunogenic, “self” antigens to a subject'simmune system in a manner that elicits an immune response powerfulenough to inhibit the growth of tumor cells in the subject. Thisinvention overcomes the previous limitations and shortcomings in the artby providing a fusion protein comprising a defensin and a tumor antigenwhich can produce an in vivo immune response, resulting in theinhibition of tumor cells. There is also a continuing need for a methodof presenting poorly antigenic viral antigens to a subject's immunesystem, particularly as relates to viral antigens such as HIV antigens.This invention also overcomes previous shortcomings in the field ofviral vaccine development by providing a fusion protein comprising adefensin and a viral antigen which is effective as a vaccine fortreating or preventing viral infection.

SUMMARY OF THE INVENTION

The present invention provides a fusion polypeptide comprising adefensin and a tumor antigen. In a preferred embodiment, the tumorantigen can be a B cell tumor antigen or MUC-1. The defensin of thisinvention can be an alpha defensin (α-def) such as human neutrophilpeptide-1 (HNP-1), human neutrophil peptide-2 (HNP-2), and/or humanneutrophil peptide-3 (HNP-3) (128), and/or a beta defensin (β-def), suchas human β-defensin-1 (HBD1), human β-defensin-2 (HBD2) (129), murineβ-defensin-2 (Def2) and/or murine β-defensin-3 (Def3).

The present invention also provides a fusion polypeptide comprising adefensin and a viral antigen. In one embodiment, the defensin can be analpha defensin (β-def) such as human neutrophil peptide-1 (HNP-1), humanneutrophil peptide-2 (HNP-2), and/or human neutrophil peptide-3 (HNP-3)(128), and/or a beta defensin (β-def), such as human β-defensin-1(HBD1), human β-defensin-2 (HBD2) (129), Def2, and/or Def3, and theviral antigen can, for example, be an HIV antigen, such as gp120, gp160,gp41, an active fragment of gp120, an active fragment of gp160 and/or anactive fragment of gp41.

In addition, the present invention provides a method of producing animmune response in a subject, comprising administering to the subjectany of the fusion polypeptides of this invention, comprising a defensinand viral antigen, or a defensin and a tumor antigen, either as aprotein or a nucleic acid encoding the fusion polypeptide.

Also provided is a method of treating a cancer in a subject comprisingadministering to the subject any of the fusion polypeptides of thisinvention, comprising a defensin and a tumor antigen, either as aprotein or a nucleic acid encoding the fusion polypeptide.

The invention also provides a method of treating or preventing a viralinfection in a subject, comprising administering to the subject any ofthe fusion polypeptides of this invention, comprising a defensin and aviral antigen, either as a protein or a nucleic acid encoding the fusionpolypeptide.

Further provided is a method of treating or preventing HIV infection ina subject, comprising administering to the subject any of the fusionpolypeptides of this invention, comprising a defensin and a humanimmunodeficiency virus (HIV) antigen, either as a protein or a nucleicacid encoding the fusion polypeptide.

A method of treating a B cell tumor in a subject is also provided,comprising administering to the subject any of the fusion polypeptidesof this invention, comprising a defensin and a B cell tumor antigen,either as a protein or a nucleic acid encoding the fusion polypeptide.

Various other objectives and advantages of the present invention willbecome apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the primary amino acid and consensus sequences for the α-and β-defensins. Boxes indicate the highly conserved cysteines that arenumbered 1 to 6. The disulfide linkages of these bridges for α-defensinsare established as 1-6, 2-4, and 3-5, whereas the disulfide linkages ofthe bridges for β-defensins are 1-5, 2-4, and 3-6. The amino acidsequences of the α-defensins HD-5 and HD-6 are not shown.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used in the claims, “a” can include multiples. For example, “a cell”can mean a single cell or more than one cell.

The present invention is based on the unexpected discovery that theadministration of a fusion protein comprising a defensin and a tumorantigen or administration of a nucleic acid encoding a fusion proteincomprising a defensin and a tumor antigen yields an effective andspecific anti-tumor immune response by converting a “self” tumor antigeninto a potent immunogen by fusing it to a defensin moiety.

Thus, the present invention provides a fusion polypeptide comprising adefensin and a tumor antigen. The fusion polypeptide can be present in apurified form and can induce an immune response against the tumorantigen and inhibit the growth of tumor cells expressing the tumorantigen. “Purified” as used herein means the polypeptide is sufficientlyfree of contaminants or cell components with which proteins normallyoccur to allow the peptide to be used therapeutically. It is notcontemplated that “purified” necessitates having a preparation that istechnically totally pure (homogeneous), but purified as used hereinmeans the fusion polypeptide is sufficiently pure to provide thepolypeptide in a state where it can be used therapeutically. As usedherein, “fusion polypeptide” means a polypeptide made up of two or moreamino acid sequences representing peptides or polypeptides fromdifferent sources. Also as used herein, “epitope” refers to a specificamino acid sequence of limited length which, when present in the properconformation, provides a reactive site for an antibody or T cellreceptor. The identification of epitopes on antigens can be carried outby immunology protocols that are standard in the art (74). As furtherused herein, “tumor antigen” describes a polypeptide expressed on thecell surface of specific tumor cells and which can serve to identify thetype of tumor. An epitope of the tumor antigen can be any site on theantigen that is reactive with an antibody or T cell receptor.

As used herein, “defensin” means an anti-microbial peptide with three tofour intramolecular cysteine disulfide bonds which induces leukocytemigration in vitro, and/or enhances concavalin A-stimulated murinespleen cell proliferation and IFN-γ production (128). As used herein, adefensin may be either a naturally occurring defensin, i.e., a peptidewhich is produced by, e.g., neutrophils, intestinal Paneth cells, orepithelial cells such as those of the skin, kidney, andtrachea-bronchial lining, or a synthetic defensin, such as a variant ofa naturally occurring defensin, which may be chemically synthesized orproduced by expressing a modified cDNA encoding a naturally occurringdefensin. While any mammalian or synthetic defensins may be used in thecompositions and methods of the invention, naturally occurring humandefensins or variants thereof are preferred.

The defensins of this invention can include, but are not limited to,alpha defensins (α-defs, such as human neutrophil peptide-1 (HNP-1),human neutrophil peptide-2 (HNP-2), human neutrophil peptide-3 (HNP-3)(128, 130), human neutrophil peptide-4 (HNP-4), human defensin-5 (HD-5),and human defensin-6 (HD-6), and beta defensins (β-defs, such as humanβ-defensin-1 (HBD1), human β-defensin-2 (HBD2) (129, 130). Humanneutrophil defensins (HNPs 1-4) are small, cationic, and arginine richpeptides that lack enzymatic activity. The peptides contain sixconserved cysteine residues that participate in 3 characteristicintramolecular disulfide bridges (130, the entire contents of thisreference is incorporated herein in its entirety). Cysteine pairingdistinguishes β-defensins from α-defensins, wherein the former areconnected 1-5, 2-4 and 3-6 and the latter are linked 1-6, 2-4 and 3-5(FIG. 1) (130). Other examples of defensins include murine β-defensin-2(Def2) and murine β-defensin-3 (Def3), as well as any other defensin nowknown or later identified.

It will be appreciated by one of skill in the art that the defensins ofthis invention can further include active fragments of defensins whichretain the activity, including chemotaxis and inhibition of chemotaxis,of the intact molecule. The production of an active fragments of adefensin and identification of defensin fragments which retain theactivity of the intact molecule are carried out according to protocolswell known in the art.

To produce a nucleic acid encoding a fusion polypeptide of thisinvention, the defensin gene can be fragmented as desired according tostandard methods and the fragments can be fused to a specific gene orgene fragment encoding an antigen to which an immune response is to beelicited (e.g., Muc-1 VNT, lymphoma scFv, gp120etc.). The fusionpolypeptide comprising the defensin fragment and the tumor or viralantigen can be produced and purified as described herein and tested forimmunogenicity according to the methods provided herein. By producingseveral fusion polypeptides having defensin fragments of varying size,the minimal size defensin fragment which imparts an immunological effectcan be identified through routine experimentation.

The tumor antigen moiety of the fusion polypeptide of this invention canbe any tumor antigen now known or later identified as a tumor antigen.The appropriate tumor antigen used in the fusion polypeptide depends onthe tumor type being treated. For example, the tumor antigen can be, butis not limited to human epithelial cell mucin (Muc-1; a 20 amino acidcore repeat for Muc-1 glycoprotein, present on breast cancer cells andpancreatic cancer cells), the Ha-ras oncogene product, p53,carcino-embryonic antigen (CEA), the raf oncogene product, GD2, GD3,GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-A/Mart-1, gp100,HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostatic serum antigen (PSA),alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras oncogeneproduct, HPV E7 and melanoma gangliosides, as well as any other tumorantigens now known or identified in the future. Tumor antigens can beobtained following known procedures or are commercially available (79).The effectiveness of the fusion protein in eliciting an immune responseagainst a particular tumor antigen can be determined according tomethods standard in the art for determining the efficacy of vaccines andaccording to the methods set forth in the Examples.

Additionally, the tumor antigen of the present invention can be anantibody which can be produced by a B cell tumor (e.g., B cell lymphoma;B cell leukemia; myeloma) or the tumor antigen can be a fragment of suchan antibody, which contains an epitope of the idiotype of the antibody.The epitope fragment can comprise as few as nine amino acids. Forexample, the tumor antigen of this invention can be a malignant B cellantigen receptor, a malignant B cell immunoglobulin idiotype, a variableregion of an immunoglobulin, a hypervariable region or complementaritydetermining region (CDR) of a variable region of an immunoglobulin, amalignant T cell receptor (TCR), a variable region of a TCR and/or ahypervariable region of a TCR.

In a preferred embodiment, the tumor antigen of this invention can be asingle chain antibody (scFv), comprising linked V_(H) and V_(L) domainsand which retains the conformation and specific binding activity of thenative idiotype of the antibody (27). Such single chain antibodies arewell known in the art and can be produced by standard methods and asdescribed in the Examples herein.

In addition, the tumor antigen of the present invention can be anepitope of the idiotype of a T cell receptor, which can be produced by aT cell tumor (e.g., T cell lymphoma; T cell leukemia; myeloma). Theepitope can comprise as few as nine amino acids.

As will be appreciated by those skilled in the art, the invention alsoincludes peptides and polypeptides having slight variations in aminoacid sequences or other properties which do not alter the functionalidentity of the peptide or polypeptide. Such variations may arisenaturally as allelic variations (e.g., due to genetic polymorphism) ormay be produced synthetically (e.g., by mutagenesis of cloned DNAsequences), such as induced point, deletion, insertion and substitutionmutations. Minor changes in amino acid sequence are generally preferred,such as conservative amino acid replacements, small internal deletionsor insertions, and additions or deletions at the ends of the molecules.Substitutions may be designed based on, for example, the model ofDayhoff et al. (80). These modifications can result in changes in theamino acid sequence, provide silent mutations, modify a restrictionsite, or provide other specific mutations while allowing for thepresentation of the functional activity of peptides and polypeptides ofthis invention.

The fusion polypeptides can comprise one or more selected epitopes onthe same tumor antigen, one or more selected epitopes on different tumorantigens, as well as repeats of the same epitope, either in tandem orinterspersed along the amino acid sequence of the fusion polypeptide.The tumor antigen can be positioned in the fusion polypeptide at thecarboxy terminus of the defensin, the amino terminus of the defensinand/or at one or more internal sites within the defensin amino acidsequence. Additionally, the fusion polypeptide can comprise more thanone defensin or active fragment thereof in any combination and in anyorder with the various tumor antigens described above.

It would be routine for an artisan to produce a fusion proteincomprising any defensin moiety and any human tumor antigen (e.g., humansingle chain antibody) moiety according to the methods described herein,on the basis of the availability in the art of the nucleic acid and/oramino acid sequence of the defensin of interest and the human tumorantigen of interest.

The present invention further provides a fusion polypeptide comprising afirst region comprising a defensin selected from the group consisting ofHNP-1, HNP-2, HNP-3, HBD1, and HBD2, and a second region comprising atumor antigen selected from the group consisting of human epithelialcell mucin (Muc-1), the Ha-ras oncogene product, p53, carcino-embryonicantigen (CEA), the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1,MAGE-3, tyrosinase, gp75, Melan-A/Mart-1, gp100, HER2/neu, EBV-LMP 1 &2, HPV-F4, 6, 7, prostatic serum antigen (PSA), alpha-fetoprotein (AFP),CO17-1A, GA733, gp72, p53, the ras oncogene product, HPV E7, proteinase3, Wilm's tumor antigen-1, telomerase, melanoma gangliosides, anantibody produced by a B cell tumor (e.g., B cell lymphoma; B cellleukemia; myeloma), a fragment of such an antibody, which contains anepitope of the idiotype of the antibody, a malignant B cell antigenreceptor, a malignant B cell immunoglobulin idiotype, a variable regionof an immunoglobulin, a hypervariable region or CDR of a variable regionof an immunoglobulin, a malignant T cell receptor (TCR), a variableregion of a TCR and/or a hypervariable region of a TCR.

For example, the present invention provides a fusion polypeptidecomprising an scFv cloned from a human subject's biopsy tumor materialor from a hybridoma cell line producing a lymphoma antibody and adefensin moiety (e.g. HNP-1, HNP-2, HNP-3, HBD1, HBD2, etc.). Inaddition, the present invention provides a defensin fused with the Muc-1core epitope of human breast cancer or human pancreatic cancer. Muc-1 isa glycoprotein (Mr>200,000) abundantly expressed on breast cancer cellsand pancreatic tumor cells. A variable number of tandem (VNT) repeats ofa 20 amino acid peptide (PDTRPAPGSTAPPAHGVTSA; SEQ ID NO:1) include Band T cell epitopes. Thus, the present invention provides a fusionprotein comprising HNP-1 and Muc-1 VNT, HNP-2 and Muc-1VNT, HNP-3 andMuc-1VNT, HBD1 and Muc-1VNT, and HBD2 and Muc-1VNT. The expressionvector is designed so that a VNT can be changed by routine cloningmethods to produce a fusion polypeptide comprising HNP-1, HNP-2, HNP-3,HBD1, or HBD2 fused with a Muc-1 VNT dimer, trimer, tetramer, pentamer,hexamer, etc.

In specific embodiments, the present invention also provides a fusionpolypeptide comprising HNP-1 and human Muc-1, HNP-2 and human Muc-1,HNP-3 and human Muc-1, HBD1 and human Muc-1, and HBD2 and human Muc-1.

The present invention further provides a fusion polypeptide comprising adefensin (e.g., HNP-1, HNP-2, HNP-3, HBD1, HBD2, etc.) and a scFv whichrecognizes tumor antigens, such as idiotype-specific scFv, Muc-1, etc.Such a fusion polypeptide would allow migration, recruitment andactivation of specialized cells of the immune system, such as naturalkiller (NK) cells, macrophages, dendritic cells (DC), polymorphonuclear(PMN) leukocytes, cytotoxic lymphocytes (CTL), etc., which would destroythe target cell.

The fusion polypeptide of this invention can further comprise a spacersequence between the defensin and the tumor antigen or viral antigen,which can have the amino acid sequence EFNDAQAPKSLE (SEQ ID NO:2), or anamino acid sequence with conservative substitutions such that it has thesame functional activity as the amino acid sequence of SEQ ID NO:2,which allows for retention of the correct folding of the tumor antigenregion of the polypeptide.

In addition, the present invention provides a composition comprising thefusion polypeptide of this invention and a suitable adjuvant. Such acomposition can be in a pharmaceutically acceptable carrier, asdescribed herein. As used herein, “suitable adjuvant” describes asubstance capable of being combined with the fusion polypeptide toenhance an immune response in a subject without deleterious effect onthe subject. A suitable adjuvant can be, but is not limited to, forexample, an immunostimulatory cytokine, SYNTEX adjuvant formulation 1(SAF-1) composed of 5 percent (wt/vol) squalene (DASF, Parsippany,N.J.), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee),and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-bufferedsaline. Other suitable adjuvants are well known in the art and includeQS-21, Freund's adjuvant (complete and incomplete), alum, aluminumphosphate, aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE) and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trealosedimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80emulsion.

The adjuvant, such as an immunostimulatory cytokine can be administeredbefore the administration of the fusion protein or nucleic acid encodingthe fusion protein, concurrent with the administration of the fusionprotein or nucleic acid or up to five days after the administration ofthe fusion polypeptide or nucleic acid to a subject. QS-21, similarly toalum, complete Freund's adjuvant, SAF, etc., can be administered withinhours of administration of the fusion protein or nucleic acid.

Furthermore, combinations of adjuvants, such as immunostimulatorycytokines can be co-administered to the subject before, after, orconcurrent with the administration of the fusion polypeptide or nucleicacid. For example, combinations of adjuvants, such as immunostimulatorycytokines, can consist of two or more of immunostimulatory cytokines ofthis invention, such as GM/CSF, interleukin-2, interleukin-12,interferon-gamma, interleukin-4, tumor necrosis factor-alpha,interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 co-stimulatorymolecules and B7.2 co-stimulatory molecules. The effectiveness of anadjuvant or combination of adjuvants may be determined by measuring theimmune response directed against the fusion polypeptide with and withoutthe adjuvant or combination of adjuvants, using standard procedures, asdescribed herein.

Furthermore, the present invention provides a composition comprising thefusion polypeptide of this invention or a nucleic acid encoding thefusion polypeptide of this invention and an adjuvant, such as animmunostimulatory cytokine or a nucleic acid encoding an adjuvant, suchas an immunostimulatory cytokine. Such a composition can be in apharmaceutically acceptable carrier, as described herein. Theimmuno-stimulatory cytokine used in this invention can be, but is notlimited to, GM/CSF, interleukin-2, interleukin-12, interferon-gamma,interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoieticfactor flt3L, CD40L, B7.1 con-stimulatory molecules and B7.2co-stimulatory molecules.

The present invention further contemplates a fusion polypeptidecomprising a defensin, or active fragment thereof, as described hereinand a viral antigen, which can be, for example, an antigen of humanimmunodeficiency virus (HIV). An HIV antigen of this invention can be,but is not limited to, the envelope glycoprotein gp120, the thirdhypervariable region of the envelope glycoprotein, gp120 of HIV-1 (thedisulfate loop V3), having the amino acid sequence:NCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNIS (SEQ ID NO:3), any otherantigenic fragment of gp120, the envelope glycoprotein gp160, anantigenic fragment of gp160, the envelope glycoprotein gp41 and/or anantigenic fragment of gp41. For example, the nucleic acid encoding theV3 loop can be fused to the 3′ end of the nucleic acid encoding adefensin (e.g., HNP-1, HNP-2, HNP-3, HBD1, or HBD2) directly orseparated by nucleic acid encoding a spacer sequence. The defensin-V3loop fusion polypeptide can be produced in an expression system asdescribed herein and purified as also described herein.

In specific embodiments, the present invention provides a fusionpolypeptide comprising a defensin and a human immunodeficiency virus(HIV) antigen, wherein the defensin can be HNP-1, HNP-2, HNP-3, HBD1,HBD2, or any other defensin, and wherein the HIV antigen can be gp120,gp160, gp41, an active (i.e., antigenic) fragment of gp120, an active(i.e., antigenic) fragment of gp160 and an active (i.e., antigenic)fragment of gp41.

Further provided in this invention is fusion polypeptide comprising anyof HNP-1 and HIV gp120, HNP-2 and HIV gp120, HNP-3 and HIV gp120, HBD1and HIV gp120, or HBD2 and HIV gp120.

An isolated nucleic acid encoding the fusion polypeptides of thisinvention as described above is also provided. By “isolated nucleicacid” is meant a nucleic acid molecule that is substantially free of theother nucleic acids and other components commonly found in associationwith nucleic acid in a cellular environment. Separation techniques forisolating nucleic acids from cells are well known in the art and includephenol extraction followed by ethanol precipitation and rapidsolubilization of cells by organic solvent or detergents (81).

The nucleic acid encoding the fusion polypeptide can be any nucleic acidthat functionally encodes the fusion polypeptide. To functionally encodethe polypeptide (i.e., allow the nucleic acid to be expressed), thenucleic acid can include, for example, expression control sequences,such as an origin of replication, a promoter, an enhancer and necessaryinformation processing sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites and transcriptional terminator sequences.Preferred expression control sequences are promoters derived frommetallothionine genes, actin genes, immunoglobulin genes, CMV, SV40,adenovirus, bovine papilloma virus, etc. A nucleic acid encoding aselected fusion polypeptide can readily be determined based upon thegenetic code for the amino acid sequence of the selected fusionpolypeptide and many nucleic acids will encode any selected fusionpolypeptide. Modifications in the nucleic acid sequence encoding thefusion polypeptide are also contemplated. Modifications that can beuseful are modifications to the sequences controlling expression of thefusion polypeptide to make production of the fusion polypeptideinducible or repressible as controlled by the appropriate inducer orrepressor. Such means are standard in the art (81). The nucleic acidscan be generated by means standard in the art, such as by recombinantnucleic acid techniques, as exemplified in the examples herein and bysynthetic nucleic acid synthesis or in vitro enzymatic synthesis.

A vector comprising any of the nucleic acids of the present inventionand a cell comprising any of the vectors of the present invention arealso provided. The vectors of the invention can be in a host (e.g., cellline or transgenic animal) that can express the fusion polypeptidecontemplated by the present invention.

There are numerous E. coli (Escherichia coli) expression vectors knownto one of ordinary skill in the art useful for the expression of nucleicacid encoding proteins such as fusion proteins. Other microbial hostssuitable for use include bacilli, such as Bacillus subtilis, and otherenterobacteria, such as Salmonella, Serratia, as well as variousPseudomonas species. These prokaryotic hosts can support expressionvectors which will typically contain expression control sequencescompatible with the host cell (e.g., an origin of replication). Inaddition, any number of a variety of well-known promoters will bepresent, such as the lactose promoter system, a tryptophan (Trp)promoter system, a beta-lactamase promoter system, or a promoter systemfrom phage lambda. The promoters will typically control expression,optionally with an operator sequence and have ribosome binding sitesequences for example, for initiating and completing transcription andtranslation. If necessary, an amino terminal methionine can be providedby insertion of a Met codon 5′ and in-frame with the protein. Also, thecarboxy-terminal extension of the protein can be removed using standardoligonucleotide mutagenesis procedures.

Additionally, yeast expression can be used. There are several advantagesto yeast expression systems. First, evidence exists that proteinsproduced in a yeast secretion system exhibit correct disulfide pairing.Second, post-translational glycosylation is efficiently carried out byyeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MFα-1 gene) isroutinely used to direct protein secretion from yeast (82). The leaderregion of pre-pro-alpha-factor contains a signal peptide and apro-segment which includes a recognition sequence for a yeast proteaseencoded by the KEX2 gene. This enzyme cleaves the precursor protein onthe carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence. Thepolypeptide coding sequence can be fused in-frame to thepre-pro-alpha-factor leader region. This construct is then put under thecontrol of a strong transcription promoter, such as the alcoholdehydrogenase I promoter or a glycolytic promoter. The protein codingsequence is followed by a translation termination codon which isfollowed by transcription termination signals. Alternatively, thepolypeptide coding sequence of interest can be fused to a second proteincoding sequence, such as Sj26 or β-galactosidase, used to facilitatepurification of the fusion protein by affinity chromatography. Theinsertion of protease cleavage sites to separate the components of thefusion protein is applicable to constructs used for expression in yeast.

Efficient post-translational glycosylation and expression of recombinantproteins can also be achieved in Baculovirus systems in insect cells.

Mammalian cells permit the expression of proteins in an environment thatfavors important post-translational modifications such as folding andcysteine pairing, addition of complex carbohydrate structures andsecretion of active protein. Vectors useful for the expression ofproteins in mammalian cells are characterized by insertion of theprotein coding sequence between a strong viral promoter and apolyadenylation signal. The vectors can contain genes conferring eithergentamicin or methotrexate resistance for use as selectable markers. Theantigen and immunoreactive fragment coding sequence can be introducedinto a Chinese hamster ovary (CHO) cell line using a methotrexateresistance-encoding vector. Presence of the vector RNA in transformedcells can be confirmed by Northern blot analysis and production of acDNA or opposite strand RNA corresponding to the protein coding sequencecan be confirmed by Southern and Northern blot analysis, respectively. Anumber of other suitable host cell lines capable of secreting intactproteins have been developed in the art and include the CHO cell lines,HeLa cells, myeloma cell lines, Jurkat cells and the like. Expressionvectors for these cells can include expression control sequences, asdescribed above.

The vectors containing the nucleic acid sequences of interest can betransferred into the host cell by well-known methods, which varydepending on the type of cell host. For example, calcium chloridetransfection is commonly utilized for prokaryotic cells, whereas calciumphosphate treatment, lipofection or electroporation may be used forother cell hosts.

Alternative vectors for the expression of protein in mammalian cells,similar to those developed for the expression of human gamma-interferon,tissue plasminogen activator, clotting Factor VIII, hepatitis B virussurface antigen, protease Nexin1, and eosinophil major basic protein,can be employed. Further, the vector can include CMV promoter sequencesand a polyadenylation signal available for expression of insertednucleic acid in mammalian cells (such as COS7).

The nucleic acid sequences can be expressed in hosts after the sequenceshave been positioned to ensure the functioning of an expression controlsequence. These expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors can contain selectionmarkers, e.g., tetracycline resistance or hygromycin resistance, topermit detection and/or selection of those cells transformed with thedesired nucleic acid sequences (83).

Additionally, the fusion polypeptides and/or nucleic acids of thepresent invention can be used in in vitro diagnostic assays, as well asin screening assays for identifying unknown tumor antigen epitopes andfine mapping of tumor antigen epitopes.

Also provided is a method for producing a fusion polypeptide comprisinga defensin, or an active fragment thereof and a tumor antigen or viralantigen, comprising cloning into an expression vector a first DNAfragment encoding a defensin or active fragment thereof and a second DNAfragment encoding a tumor antigen or viral antigen; and expressing theDNA of the expression vector in an expression system under conditionswhereby the fusion polypeptide is produced. The expression vector andexpression system can be of any of the types as described herein. Thecloning of the first and second DNA segments into the expression vectorand expression of the DNA under conditions which allow for theproduction of the fusion protein of this invention can be carried out asdescribed in the Examples section included herein. The method of thisinvention can further comprise the step of isolating and purifying thefusion polypeptide, according to methods well known in the art and asdescribed herein.

Any of the fusion polypeptides, the nucleic acids and the vectors of thepresent invention can be in a pharmaceutically acceptable carrier and inaddition, can include other medicinal agents, pharmaceutical agents,carriers, diluents, adjuvants (e.g., immunostimulatory cytokines), etc.By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected antigen withoutcausing substantial deleterious biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Actual methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art (84).

Thus, the present invention further provides a method for inducing animmune response in a subject capable of induction of an immune responseand preferably human, comprising administering to the subject an immuneresponse-inducing amount of the fusion polypeptide of this invention. Asused herein, “an immune response-inducing amount” is that amount offusion polypeptide which is capable of producing in a subject a humoraland/or cellular immune response capable of being detected by standardmethods of measurement, such as, for example, as described herein. Forexample, the antigenic polypeptide region can induce an antibodyresponse. The antibodies can treat or prevent a pathological or harmfulcondition in the subject in which the antibodies are produced or theantibodies can be removed from the subject and administered to anothersubject to treat or prevent a pathological or harmful condition. Thefusion polypeptide can also induce an effector T cell (cellular) immuneresponse which is effective in treating or preventing a pathological orharmful conditions in the subject.

In an embodiment wherein the antigen moiety of the fusion polypeptidecomprises an immunoglobulin light or heavy chain or a single chainantibody, the immune response can be the production in the subject ofanti-idiotype antibodies, which represent the image of the originalantigen and can function in a vaccine preparation to induce an immuneresponse to a pathogenic antigen, thereby avoiding immunization with theantigen itself (85). The anti-idiotype antibodies can treat or prevent apathological or harmful condition in the subject in which theanti-idiotype antibodies are produced or the anti-idiotype antibodiescan be removed from the subject and administered to another subject totreat or prevent a pathological or harmful condition.

Further provided is a method for inhibiting the growth of tumor cells ina subject, comprising administering to the subject a tumor cellgrowth-inhibiting amount of the fusion polypeptide of this invention.The subject of this method can be any subject in which a humoral and/orcellular immune response to a tumor can be induced, which is preferablyan animal and most preferably a human. As used herein, “inhibiting thegrowth of tumor cells” means that, following administration of thefusion polypeptide, a measurable humoral and/or cellular immune responseagainst the tumor cell epitope is elicited in the subject, resulting inthe inhibition of growth of tumor cells present in the subject. Thehumoral immune response can be measured by detection, in the serum ofthe subject, of antibodies reactive with the epitope of the tumorantigen present on the fusion polypeptide, according to protocolsstandard in the art, such as enzyme linked immunosorbent immunoassay(ELISA) and Western blotting protocols. The cellular immune response canbe measured by, for example, footpad swelling in laboratory animals,peripheral blood lymphocyte (PBL) proliferation assays and PBLcytotoxicity assays, as would be known to one of ordinary skill in theart of immunology and particularly as set forth in the availablehandbooks and texts of immunology protocols (86).

The present invention also provides a method of treating cancer in asubject diagnosed with cancer, comprising administering to the subjectan effective amount of the fusion polypeptide of the present invention.The cancer can be, but is not limited to B cell lymphoma, T celllymphoma, myeloma, leukemia, breast cancer, pancreatic cancer, coloncancer, lung cancer, renal cancer, liver cancer, prostate cancer,melanoma and cervical cancer.

Further provided is a method of treating a B cell tumor in a subjectdiagnosed with a B cell tumor, comprising administering an effectiveamount of the fusion polypeptide of this invention, which comprises anantibody or a fragment thereof, as described herein, in apharmaceutically acceptable carrier, to the subject.

In specific embodiments, the present invention also provides a method ofproducing an immune response in a subject, comprising administering tothe subject a composition comprising a fusion polypeptide of thisinvention and a pharmaceutically acceptable carrier and wherein thefusion polypeptide can be a fusion polypeptide comprising any of HNP-1and human Muc-1, HNP-2 and human Muc-1, HNP-3 and human Muc-1, HBD1 andhuman Muc-1, HBD2 and human Muc-1, Def2 and human Muc-1, and Def3 andhuman Muc-1.

Also provided is a method of producing an immune response in a subject,comprising administering to the subject a composition comprising anucleic acid encoding a fusion polypeptide of this invention and apharmaceutically acceptable carrier and wherein the fusion polypeptideis a fusion polypeptide comprising any of HNP-1 and human Muc-1, HNP-2and human Muc-1, HNP-3 and human Muc-1, HBD1 and human Muc-1, HBD2 andhuman Muc-1, Def2 and human Muc-1, and Def3 and human Muc-1, underconditions whereby the nucleic acid of the composition can be expressed,thereby producing an immune response in the subject.

In further embodiments, the present invention also provides a method ofproducing an immune response in a subject, comprising administering tothe subject a composition comprising a fusion polypeptide of thisinvention and a pharmaceutically acceptable carrier and wherein thefusion polypeptide can be a fusion polypeptide comprising any of HNP-1and HIV gp120, HNP-2 and HIV gp120, HNP-3 and HIV gp120, HBD1 and HIVgp120, HBD2 and HIV gp120, Def2 and HIV gp120, or Def3 and HIV gp120,thereby producing an immune response in the subject.

Also provided is a method of producing an immune response in a subject,comprising administering to the subject a composition comprising anucleic acid encoding a fusion polypeptide of this invention and apharmaceutically acceptable carrier and wherein the fusion polypeptideis a fusion polypeptide comprising any of HNP-1 and HIV gp120, HNP-2 andHIV gp120, HNP-3 and HIV gp120, HBD1 and HIV gp120, HBD2 and HIV gp120,Def2 and HIV gp120, or Def3 and HIV gp120, under conditions whereby thenucleic acid of the composition can be expressed, thereby producing animmune response in the subject.

Also provided is a method of producing an immune response in a subject,comprising administering to the subject a composition comprising afusion polypeptide and a pharmaceutically acceptable carrier and whereinthe fusion polypeptide is a fusion polypeptide comprising a defensin anda human immunodeficiency virus (HIV) antigen, wherein the defensin canbe HNP-1, HNP-2, HNP-3, HBD1, HBD2, Def2, Def3, or any other defensin,and wherein the HIV antigen can be gp120, gp160, gp41, an active (i.e.,antigenic) fragment of gp120, an active (i.e., antigenic) fragment ofgp160 and/or an active (i.e., antigenic) fragment of gp41, therebyproducing an immune response in the subject.

The present invention also provides a method of producing an immuneresponse in a subject, comprising administering to the subject acomposition comprising a nucleic acid encoding a fusion polypeptidecomprising a defensin and a human immunodeficiency virus (HIV) antigen,wherein the defensin can be HNP-1, HNP-2, HNP-3, HBD1, HBD2, Def2, Def3,or any other defensin, and wherein the HIV antigen can be gp120, gp160,gp41, an active (i.e., antigenic) fragment of gp120, an active (i.e.,antigenic) fragment of gp160 and/or an active (i.e., antigenic) fragmentof gp41, and a pharmaceutically acceptable carrier, under conditionswhereby the nucleic acid can be expressed, thereby producing an immuneresponse in the subject.

In any of the methods provided herein which recite the production of animmune response, the immune response can be humoral and/or an effector Tcell (cellular) immune response, as determined according to methodsstandard in the art.

In another embodiment, the present invention provides a method oftreating a cancer in a subject comprising administering to the subject acomposition comprising a fusion polypeptide of this invention and apharmaceutically acceptable carrier and wherein the fusion polypeptideis a fusion polypeptide comprising any of HNP-1 and human Muc-1, HNP-2and human Muc-1, HNP-3 and human Muc-1, HBD1 and human Muc-1, HBD2 andhuman Muc-1, Def2 and human Muc-1, or Def3 and human Muc-1, therebytreating a cancer in the subject.

Additionally provided is a method of treating a cancer in a subject,comprising administering to the subject a composition comprising anucleic acid encoding a fusion polypeptide of this invention and apharmaceutically acceptable carrier and wherein the fusion polypeptideis a fusion polypeptide comprising any of HNP-1 and human Muc-1, HNP-2and human Muc-1, HNP-3 and human Muc-1, HBD1 and human Muc-1, HBD2 andhuman Muc-1, Def2 and human Muc-1, or Def3 and human Muc-1, underconditions whereby the nucleic acid of the composition can be expressed,thereby treating a cancer in the subject.

Further provided is a method of treating or preventing HIV infection ina subject, comprising administering to the subject a compositioncomprising a defensin and a human immunodeficiency virus (HIV) antigen,wherein the defensin can be HNP-1, HNP-2, HNP-3, HBD1, HBD2, Def2, Def3,or any other defensin, and wherein the HIV antigen can be gp120, gp160,gp41, an active (i.e., antigenic) fragment of gp120, an active (i.e.,antigenic) fragment of gp160 and/or an active (i.e., antigenic) fragmentof gp41, and a pharmaceutically acceptable carrier, thereby treating orpreventing HIV infection in the subject.

In addition, a method of treating or preventing HIV infection in asubject is provided herein, comprising administering to the subject acomposition comprising a nucleic acid encoding a fusion polypeptidecomprising a defensin and a human immunodeficiency virus (HIV) antigen,wherein the defensin can be HNP-1, HNP-2, HNP-3, HBD1, HBD2, Def2, Def3,or any other defensin, and wherein the HIV antigen can be gp120, gp160,gp41, an active (i.e., antigenic) fragment of gp120, an active (i.e.,antigenic) fragment of gp160 and/or an active (i.e., antigenic) fragmentof gp41, and a pharmaceutically acceptable carrier, under conditionswhereby the nucleic acid can be expressed, thereby treating orpreventing HIV infection in the subject.

Further provided is a method of treating or preventing HIV infection ina subject, comprising administering to the subject a compositioncomprising a fusion polypeptide comprising any of HNP-1 and HIV gp120,HNP-2 and HIV gp120, HNP-3 and HIV gp120, HBD1 and HIV gp120, HBD2 andHIV gp120, Def2 and V gp120, or Def3 and HIV gp120, and apharmaceutically acceptable carrier, thereby treating or preventing HIVinfection in the subject.

In addition, a method of treating or preventing HIV infection in asubject is provided herein, comprising administering to the subject acomposition comprising a nucleic acid encoding a fusion polypeptidecomprising any of HNP-1 and HIV gp120, HNP-2 and HIV gp120, HNP-3 andHIV gp120, HBD1 and HIV gp120, gp120, HBD2 and HIV gp120, Def2 and HIVgp120, or Def3 and HIV gp120, and a pharmaceutically acceptable carrier,under conditions whereby the nucleic acid can be expressed, therebytreating or preventing HIV infection in the subject.

In a further embodiment, the present invention provides a method oftreating a B cell tumor in a subject, comprising administering to thesubject a fusion polypeptide comprising a defensin and a B cell tumorantigen, wherein the B cell tumor antigen can be an antibody, a singlechain antibody or an epitope of an idiotype of an antibody, and whereinthe defensin can be HNP-1, HNP-2, HNP-3, HBD1, HBD2, Def2, Def3, or anyother defensin, thereby treating a B cell tumor in the subject.

Also provided is a fusion polypeptide comprising the defensin HNP-1,HNP-2, HNP-3, HBD1, HBD2, Def2, Def3, or any other defensin, and the V3loop of HIV-1 envelope glycoprotein, gp120, as well as a fusion proteincomprising HNP-1, HNP-2, HNP-3, HBD1, HBD2, Def2, Def3, or any otherdefensin, and gp160 of HIV-1, a fusion protein comprising HNP-1, HNP-2,HNP-3, HBD1, HBD2, def2, Def3, or any other defensin, and gp41 of HIV-1,a fusion protein comprising HNP-1, HNP-2, HNP-3, HBD1, HBD2, Def2, Def3,or any other defensin, and an active fragment of gp120, a fusion proteincomprising HNP-1, HNP-2, HNP-3, HBD1, HBD2, Def2, Def3, or any otherdefensin, and an active fragment of gp160 and a fusion polypeptidecomprising HNP-1, HNP-2, HNP-3, HBD1, HBD2, Def2, Def3, or any otherdefensin, and an active fragment of gp41.

The methods of this invention comprising administering the fusionprotein of this invention to a subject can further comprise the step ofadministering one or more adjuvants, such as an immunostimulatorycytokine to the subject. The adjuvant or adjuvants can be administeredto the subject prior to, concurrent with and/or after the administrationof the fusion protein as described herein.

The subject of the present invention can be any animal in which cancercan be treated by eliciting an immune response to a tumor antigen. In apreferred embodiment, the animal is a mammal and most preferably is ahuman.

To determine the effect of the administration of the fusion polypeptideon inhibition of tumor cell growth in laboratory animals, the animalscan either be pre-treated with the fusion polypeptide and thenchallenged with a lethal dose of tumor cells, or the lethal dose oftumor cells can be administered to the animal prior to receipt of thefusion polypeptide and survival times documented. To determine theeffect of administration of the fusion polypeptide on inhibition oftumor cell growth in humans, standard clinical response parameters canbe analyzed.

To determine the amount of fusion polypeptide which would be aneffective tumor cell growth-inhibiting amount, animals can be treatedwith tumor cells as described herein and varying amounts of the fusionpolypeptide can be administered to the animals. Standard clinicalparameters, as described herein, can be measured and that amount offusion polypeptide effective in inhibiting tumor cell growth can bedetermined. These parameters, as would be known to one of ordinary skillin the art of oncology and tumor biology, can include, but are notlimited to, physical examination of the subject, measurements of tumorsize, X-ray studies and biopsies.

The present invention further provides a method for treating orpreventing HIV infection in a human subject, comprising administering tothe subject an HIV replication-inhibiting amount of the defensin/HIVantigen fusion polypeptide of this invention. As used herein, “areplication-inhibiting amount” is that amount of fusion polypeptidewhich produces a measurable humoral and/or effector T cell (cellular)immune response in the subject against the viral antigen, as determinedby standard immunological protocols, resulting in the inhibition of HIVreplication in cells of the subject, as determined by methods well knownin the art for measuring HIV replication, such as viral loadmeasurement, which can be determined by quantitative PCR (QPCR) andbranched DNA (bDNA) analysis; reverse transcriptase activitymeasurement, in situ hybridization, Western immunoblot, ELISA and p24gag measurement (87, 88, 89, 90, 91). The fusion polypeptide can beadministered to the subject in varying amounts and the amount of thefusion polypeptide optimally effective in inhibiting HIV replication ina given subject can be determined as described herein.

The fusion polypeptide of this invention can be administered to thesubject orally or parenterally, as for example, by intramuscularinjection, by intraperitoneal injection, topically, transdermally,injection directly into the tumor, or the like, although subcutaneousinjection is typically preferred. Immunogenic, tumor cell growthinhibiting and HIV replication inhibiting amounts of the fusionpolypeptide can be determined using standard procedures, as described.Briefly, various doses of the fusion polypeptide are prepared,administered to a subject and the immunological response to each dose isdetermined (92). The exact dosage of the fusion polypeptide will varyfrom subject to subject, depending on the species, age, weight andgeneral condition of the subject, the severity of the cancer or HIVinfection that is being treated, the particular antigen being used, themode of administration, and the like. Thus, it is not possible tospecify an exact amount. However, an appropriate amount may bedetermined by one of ordinary skill in the art using only routinescreening given the teachings herein.

Generally, the dosage of fusion protein will approximate that which istypical for the administration of vaccines, and typically, the dosagewill be in the range of about 1 to 500 μg of the fusion polypeptide perdose, and preferably in the range of 50 to 250 μg of the fusionpolypeptide per dose. This amount can be administered to the subjectonce every other week for about eight weeks or once every other monthfor about six months. The effects of the administration of the fusionpolypeptide can be determined starting within the first month followingthe initial administration and continued thereafter at regularintervals, as needed, for an indefinite period of time.

For oral administration of the fusion polypeptide of this invention,fine powders or granules may contain diluting, dispersing, and/orsurface active agents, and may be presented in water or in a syrup, incapsules or sachets in the dry state, or in a nonaqueous solution orsuspension wherein suspending agents may be included, in tablets whereinbinders and lubricants may be included, or in a suspension in water or asyrup. Where desirable or necessary, flavoring, preserving, suspending,thickening, or emulsifying agents may be included. Tablets and granulesare preferred oral administration forms, and these may be coated.

Parenteral administration, if used, is generally characterized byinjection. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. A morerecently revised approach for parenteral administration involves use ofa slow release or sustained release system, such that a constant levelof dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which isincorporated by reference herein.

For solid compositions, conventional nontoxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose,magnesium carbonate, and the like. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving, dispersing,etc. an active compound as described herein and optional pharmaceuticaladjuvants in an excipient, such as, for example, water, saline, aqueousdextrose, glycerol, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, etc. Actualmethods of preparing such dosage forms are known, or will be apparent,to those skilled in this (84).

The present invention also provides a method for producing single chainantibodies against tumor antigens comprising producing a fusionpolypeptide comprising a defensin region and a region comprising a tumorantigen; immunizing animals with an amount of the fusion polypeptidesufficient to produce a humoral immune response to the fusionpolypeptide; isolating spleen cells expressing immunoglobulin specificfor the fusion polypeptide; isolating the immunoglobulin variable genesfrom the spleen cells; cloning the immunoglobulin variable genes into anexpression vector; expressing the immunoglobulin variable genes in abacteriophage; infecting E. coli cells with the bacteriophage; isolatingbacteriophage from the E. coli cells which express the immunoglobulinvariable genes and isolating the immunoglobulin variable gene productsfor use as single chain antibodies.

The defensin-scFv fusion proteins described herein would be bettertargets than tumor cells or purified tumor antigen peptides for antibodyselection approaches such as phage displayed scFv production. Forexample, there are two ways to produce specific Fv displayed on thesurface of phage: (1) Immunize mice with tumor cells; isolateimmunoglobulin variable fragment genes from spleen cells by RT/PCR;clone the genes into bacteriophage in frame with genes coding phagesurface proteins (e.g., major coat protein subunits gp VIII or gp III ofthe filamentous bacteriophage) (93, 94); and (2) Construct semisyntheticantibody libraries by PCR as described (95). The specific phageproducing scFv are selected by several rounds of binding elution andinfection in E. coli, using biotin labeled defensin-tumor antigen (e.g.,Muccore). The biotin enables selection of high affinity scFv-phagethrough binding to streptavidin conjugated magnetic beads. This approachprovides simple, fast and efficient production of specific anti-tumorepitope scFv.

As described herein, the present invention also provides a nucleic acidwhich encodes a fusion polypeptide of this invention and a vectorcomprising a nucleic acid which encodes a fusion polypeptide of thisinvention, either of which can be in a pharmaceutically acceptablecarrier. Such nucleic acids and vectors can be used in gene therapyprotocols to treat cancer as well as to treat or prevent HIV infectionin a subject.

Thus, the present invention further provides a method of treating acancer in a subject diagnosed with a cancer comprising administering thenucleic acid of this invention to a cell of the subject under conditionswhereby the nucleic acid is expressed in the cell, thereby treating thecancer.

A method of treating a B cell tumor in a subject diagnosed with a B celltumor is also provided, comprising administering the nucleic acid ofthis invention, encoding a defensin and an antibody or fragment thereof,in a pharmaceutically acceptable carrier, to a cell of the subject,under conditions whereby the nucleic acid is expressed in the cell,thereby treating the B cell tumor.

The methods of this invention comprising administering nucleic acidencoding the fusion protein of this invention to a subject can furthercomprise the step of administering a nucleic acid encoding an adjuvantsuch as an immunostimulatory cytokine to the subject, either before,concurrent with or after the administration of the nucleic acid encodingthe fusion protein, as described herein.

The nucleic acid can be administered to the cell in a virus, which canbe, for example, adenovirus, retrovirus and adeno-associated virus.Alternatively, the nucleic acid of this invention can be administered tothe cell in a liposome. The cell of the subject can be either in vivo orex vivo. Also, the cell of the subject can be any cell which can take upand express exogenous nucleic acid and produce the fusion polypeptide ofthis invention. Thus, the fusion polypeptide of this invention can beproduced by a cell which secretes it, whereby it binds a defensinreceptor and is subsequently processed by an antigen presenting cell andpresented to the immune system for elicitation of an immune response.Alternatively, the fusion polypeptide of this invention can be producedin an antigen presenting cell where it is processed directly andpresented to the immune system.

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The nucleic acids of this invention can be introduced intothe cells via any gene transfer mechanism, such as, for example,virus-mediated gene delivery, calcium phosphate mediated gene delivery,electroporation, microinjection or proteoliposomes. The transduced cellscan then be infused (e.g., in a pharmaceutically acceptable carrier) ortransplanted back into the subject per standard methods for the cell ortissue type. Standard methods are known for transplantation or infusionof various cells into a subject.

For in vivo methods, the nucleic acid encoding the fusion protein can beadministered to the subject in a pharmaceutically acceptable carrier asdescribed herein.

In the methods described herein which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids of the presentinvention can be in the form of naked DNA or the nucleic acids can be ina vector for delivering the nucleic acids to the cells for expression ofthe nucleic acid to produce the fusion protein of this invention. Thevector can be a commercially available preparation, such as anadenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec,Canada). Delivery of the nucleic acid or vector to cells can be via avariety of mechanisms. As one example, delivery can be via a liposome,using commercially available liposome preparations such as LIPOFECTIN,LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen,Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,Wis.), as well as other liposomes developed according to proceduresstandard in the art. In addition, the nucleic acid or vector of thisinvention can be delivered in vivo by electroporation, the technologyfor which is available from Genetronics, Inc. (San Diego, Calif.) aswell as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp.,Tucson, Ariz.).

Vector delivery can also be via a viral system, such as a retroviralvector system which can package a recombinant retroviral genome (seee.g., 96, 97). The recombinant retrovirus can then be used to infect andthereby deliver to the infected cells nucleic acid encoding the fusionpolypeptide. The exact method of introducing the exogenous nucleic acidinto mammalian cells is, of course, not limited to the use of retroviralvectors. Other techniques are widely available for this procedureincluding the use of adenoviral vectors (98), adeno-associated viral(AAV) vectors (99), lentiviral vectors (100), pseudotyped retroviralvectors (101). Physical transduction techniques can also be used, suchas liposome delivery and receptor-mediated and other endocytosismechanisms (see, for example, 102). This invention can be used inconjunction with any of these or other commonly used gene transfermethods.

Various adenoviruses may be used in the compositions and methodsdescribed herein. For example, a nucleic acid encoding the fusionprotein can be inserted within the genome of adenovirus type 5.Similarly, other types of adenovirus may be used such as type 1, type 2,etc. For an exemplary list of the adenoviruses known to be able toinfect human cells and which therefore can be used in the presentinvention, see Fields, et al. (103). Furthermore, it is contemplatedthat a recombinant nucleic acid comprising an adenoviral nucleic acidfrom one type adenovirus can be packaged using capsid proteins from adifferent type adenovirus.

The adenovirus of the present invention is preferably renderedreplication deficient, depending upon the specific application of thecompounds and methods described herein. Methods of rendering anadenovirus replication deficient are well known in the art. For example,mutations such as point mutations, deletions, insertions andcombinations thereof, can be directed toward a specific adenoviral geneor genes, such as the E1 gene. For a specific example of the generationof a replication deficient adenovirus for use in gene therapy, see WO94/28938 (Adenovirus Vectors for Gene Therapy Sponsorship) which isincorporated herein in its entirety.

In the present invention, the nucleic acid encoding the fusion proteincan be inserted within an adenoviral genome and the fusion proteinencoding sequence can be positioned such that an adenovirus promoter isoperatively linked to the fusion protein nucleic acid insert such thatthe adenoviral promoter can then direct transcription of the nucleicacid, or the fusion protein insert may contain its own adenoviralpromoter. Similarly, the fusion protein insert may be positioned suchthat the nucleic acid encoding the fusion protein may use otheradenoviral regulatory regions or sites such as splice junctions andpolyadenylation signals and/or sites. Alternatively, the nucleic acidencoding the fusion protein may contain a different enhancer/promoter(e.g., CMV or RSV-LTR enhancer/promoter sequences) or other regulatorysequences, such as splice sites and polyadenylation sequences, such thatthe nucleic acid encoding the fusion protein may contain those sequencesnecessary for expression of the fusion protein and not partially ortotally require these regulatory regions and/or sites of the adenovirusgenome. These regulatory sites may also be derived from another source,such as a virus other than adenovirus. For example, a polyadenylationsignal from SV40 or BGH may be used rather than an adenovirus, a human,or a murine polyadenylation signal. The fusion protein nucleic acidinsert may, alternatively, contain some sequences necessary forexpression of the nucleic acid encoding the fusion protein and deriveother sequences necessary for the expression of the fusion proteinnucleic acid from the adenovirus genome, or even from the host in whichthe recombinant adenovirus is introduced.

As another example, for administration of nucleic acid encoding thefusion protein to an individual in an AAV vector, the AAV particle canbe directly injected intravenously. The AAV has a broad host range, sothe vector can be used to transduce any of several cell types, butpreferably cells in those organs that are well perfused with bloodvessels. To more specifically administer the vector, the AAV particlecan be directly injected into a target organ, such as muscle, liver orkidney. Furthermore, the vector can be administered intraarterially,directly into a body cavity, such as intraperitoneally, or directly intothe central nervous system (CNS).

An AAV vector can also be administered in gene therapy procedures invarious other formulations in which the vector plasmid is administeredafter incorporation into other delivery systems such as liposomes orsystems designed to target cells by receptor-mediated or otherendocytosis procedures. The AAV vector can also be incorporated into anadenovirus, retrovirus or other virus which can be used as the deliveryvehicle.

As described above, the nucleic acid or vector of the present inventioncan be administered in vivo in a pharmaceutically acceptable carrier. By“pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject, along with the nucleic acid or vector,without causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier wouldnaturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

The mode of administration of the nucleic acid or vector of the presentinvention can vary predictably according to the disease being treatedand the tissue being targeted. For example, for administration of thenucleic acid or vector in a liposome, catheterization of an arteryupstream from the target organ is a preferred mode of delivery, becauseit avoids significant clearance of the liposome by the lung and liver.

The nucleic acid or vector may be administered orally as describedherein for oral administration of the fusion polypeptides of thisinvention, parenterally (e.g., intravenously), by intramuscularinjection, by intraperitoneal injection, transdermally,extracorporeally, topically or the like, although intravenousadministration is typically preferred. The exact amount of the nucleicacid or vector required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the disorder being treated, the particular nucleic acid orvector used, its mode of administration and the like. Thus, it is notpossible to specify an exact amount for every nucleic acid or vector.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein (84).

As one example, if the nucleic acid of this invention is delivered tothe cells of a subject in an adenovirus vector, the dosage foradministration of adenovirus to humans can range from about 10⁷ to 10⁹plaque forming units (pfu) per injection, but can be as high as 10¹² pfuper injection (104, 105). Ideally, a subject will receive a singleinjection. If additional injections are necessary, they can be repeatedat six month intervals for an indefinite period and/or until theefficacy of the treatment has been established.

Parenteral administration of the nucleic acid or vector of the presentinvention, if used, is generally characterized by injection. Injectablescan be prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. A more recently revised approachfor parenteral administration involves use of a slow release orsustained release system such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference hereinin its entirety.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES Example 1

Materials and Methods

Mice and tumor. C3H/HeN female mice 6 to 12 weeks of age are obtainedfrom the Animal Production Area of the National CancerInstitute-Frederick Cancer Research and Development Center (NCI-FCRDC,Frederick, Md.). The cell line 38c13 is a carcinogen-induced murine Bcell tumor cell line (125). The 38c13 tumor cell secretes and expressesIgM(κ) on the cell surface and is MHC class I positive but class IInegative. 38c13 cells from a common frozen stock are passaged in vitro 3days before use in RPMI 1640 supplemented with 100 U/ml of penicillinand streptomycin, 2×10⁻⁵M 2-mercaptoethanol and heat inactivated 10%fetal bovine serum (BioWhitaker).

Construction of expression vectors. Two types of expression systems areused to produce scFv and scFv fusions. In one system, nucleic acidencoding the fusion protein is expressed in a modified pet11d vector(Stratagene) and purified from inclusion bodies of E. coli. In thesecond system, the nucleic acid encoding the fusion polypeptide iscloned into a pCMVE/AB (Arya Biragyn) vector under regulatory elementsof the early promoter and enhancer of CMV and expressed in the epidermisof mice as a naked DNA vaccine.

Fv fragments are cloned from two different B cell lymphomas, 38C13 andA20, respectively (106, 107) by RT/PCR and produced as recombinantfusion peptides with either IP-10, respectively designated as IP10scFv38and IP10scFv20A, or MCP3scFv38 and MCP3scFv20A. Specifically, lymphomaspecific Vh and Vl fragments are cloned by RT/PCR techniques as singlechain antibody from total RNA of 38c13 and A20 tumor cells, designatedscFv38 and scFv20A respectively, using the following primers.

PRVh-5′: PRV_(H)38-5′: CTCGAGG TGAAGCTGGTGGAGTCTGGA (SEQ ID NO: 4)PRVh-3′: PRV_(H)38-3′: AGAGGAGA CTGTGAGAGTGGTGCCTT (SEQ ID NO: 5)PRV1-5′: PRV_(L)38-5′: GACATCCAGATGACACAGTCTCCA (SEQ ID NO: 6) PRV1-3′:PRV_(L)38-3′: GGATCCTTTTATTTCCAGCTTGGTCCCCCCTCCGAA (SEQ ID NO: 7)PRV_(H)20A-5′: CCATGGTCCAAC TGCAGCAGTCAGGGCCTGAC (SEQ ID NO: 8)PRV_(H)20A-3′: TGAGGAGACTGTGAGTTCGGTACCTT GGCC (SEQ ID NO: 9)PRV_(L)20A-5′: GATGTTGTGATGACGCAGACTCCACTC (SEQ ID NO: 10)pRV_(L)20A-3′: GGATCCTT TGACTTCCAGCTTTGTGCCTCCA (SEQ ID NO: 11)

The resulting scFv contained a (Gly₄Ser)₃ linker and is cloned into theexpression vector pET11d, which is modified to fuse in frame with c-mycand the His tag peptide sequences, followed by an amber stop codon. Theresulting scFv contains a 17 a.a. residue linker, GGGGSGGGGSGGGGSGS(Gly₄Ser)₃GlySer (SEQ ID NO:12) (108).

Constructs for the nDNA vaccination are fused in frame to the followingdefensins lacking the pro-sequence, as defensins are first produced asinactive pro-defensins, and are activated by proteolytic cleavage of thepro sequence. Thus, the nucleic acids encoding murine β defensin-2(Def2), murine β defensin-3(Def3), HNP-1, HNP-2, HNP-3, HBD1, or HBD2lacking their pro sequence are inserted into pCMVE/AB to enablesecretion. The carboxy-terminus of scFv is then fused in frame with thetag sequence encoding c-myc peptide and six His residues, respectively:GGA TCC GCA GAA GAA CAG AAA CTG ATC TCA GAA GAG GAT CTG GCC CAC CAC CATCAC CAT CAC TAA CCCGGG (SEQ ID NO:13). Genes for the mature sequence(i.e., lacking the pro-sequence) of defensins HNP-1, HNP-2, HNP-3, HBD1,and HBD2, are cloned by RT/PCR technique from cell lines which are knownin the art, and fused in frame with sFv utilizing suitable primers toform IP-1sFv38, HNP-2sFv38, HNP-3sFv38, HBD1sFv38, and HBD2sFv38. Murinebeta defensin-2 and -3 are cloned by RT/PCR from the LPS-treated murineskin and fused in frame with model lymphoma derived sFv antigen, selftumor antigen (Def2sFv38 and Def3sFv38, respectively). As controls, sFvfusion proteins with the pro-defensin form of each of the above listeddefensins are prepared.

Def2, Def3, HNP-1, HNP-2, HNP-3, HBD1, and HBD2 fusions are made byfusing them to amino-terminus of scFv through a short spacer sequence:5′ GAA TTC AAC GAC GCT CAG GCG CCG AAG AGT CTC GAG 3′ (SEQ ID NO:14),encoding the amino acid sequence: EFNDQAPKSLE (SEQ ID NO:15). Two uniquerestriction endonuclease sites are introduced at the ends of the spaceto facilitate cloning: EcoRI at the 5′ end (underlined) and XhoI at the3′ end (underlined). All constructs are verified by DNAdideoxy-sequencing method, using T7 SEQUENASE kit (Amersham).

Def2, Def3, HNP-1, HNP-2, HNP-3, HBD1, and HBD2 defensins are clonedinto the scFv38 expression vector through NcoI and XhoI restrictionsites. The resulting fusion nucleic acid contains the defensin geneligated to the 5′-end of the scFv38 gene and separated with a shortspacer sequence, as described above.

Bacterial expression and scFv purification. The recombinant proteins areexpressed in BL21(DE3) cells (InVitrogen) as inclusion bodies after 8hours of induction in Super-Broth with 0.8 mM IPTG in the presence of150 μg/ml carbenicillin and 50 μg/ml ampicillin at 30° C. HNP-1-scFv38,HNP-2-scFv38, HNP-3-scFv38, HBD1-scFv38, and HBD2-scFv38, are purifiedfrom the inclusion bodies with a modified method (110). Briefly,inclusion bodies, denatured in 6M Gu HCl, 100 mM NaH₂PO₄, 10 mMTris-HCl, pH 8.0, are reduced in 0.3M DTE and refolded at aconcentration of 80 μg/ml in the refolding solution (Tris-HCl, pH 8.0,0.5M arginine-HCL, 4 mM GSSG and 2 mM EDTA) for 72 hours at 10° C. Therefolded solution is dialyzed in 100 mM Urea and 20 mM tris-HCl, pH 7.4and the recombinant protein is purified by binding to heparin-sepharoseresins (Pharmacia, Biotech, Uppsala, Sweden). The integrity and purityof the recombinant protein is tested by PAGE gel electrophoresis inreducing conditions and by Western blot hybridization with mAb 9E10. Thepurification yields 2-20 mg/l of the soluble protein with greater than90% purity.

Results

Purified fusion polypeptide is tested for the ability to inhibit bindingof native IgM 38c13 (Id38), as compared to positive sera from miceimmunized with Id38-KLH. ELISA plates are coated with 10 μg/ml Id38,then wells are incubated with anti-Id38 positive sera (1:500) andtitrated amounts of scFv. Id38 (10 μg/ml) and either Def2, Def3, HNP-1scFv20, HNP-2 scFv20, HNP-3 scFv20, HBD1 scFv20, or HBD2 scFv20 HNP-1,HNP-2, HNP-3, HBD1, and HBD2 defensins fused to an irrelevant scFv) areused as positive and negative control samples, respectively.

Recombinant fusion proteins purified from E. coli are characterized forproper idiotype folding by their ability to inhibit 38c13 IgM binding toa monoclonal (SIC5 mAb) or polyclonal anti-idiotypic sera, in order todetermine if Def2, Def3, HNP-1, HNP-2, HNP-3, HBD1, and HBD2 defensinfusions interfere with the proper conformation of scFv38.

Next, defensin fusions, including controls, are tested for their abilityto induce chemotaxis of different subsets of immune cells, includingspenocytes and bone marrow derived immature dendritic cells. BothDef2sFv38 and Def3sFv38 induce chemotaxis of murine splenocytes andmurine bone marrow derived immature DC in dose dependent manner with themaximum migration to 10 ng/ml and 100 ng/ml of Def2sFv38 and Def3sFv38,respectively. The immature phenotype of these DC has been confirmed byFacs staining and by supported by their ability to migrate to MIP3, achemo-attractant specific for immature DC, and not react to MIP3, matureDC chemo-attractant. In contrast, no chemotaxis is detected when cellsincubated with proDef2sFv38 or control protein MCP3TsFv38. Moreover, dayseven bone marrow derived DC, which acquired mature phenotype as judgedby Facs and by inability to migrate to MIP3 while acquiring migration toMIP3, do not respond to either of defensin fusions. Therefore, murinebeta defensin-2 and -3 fusion proteins target specifically immature DCinducing their chemotaxis, and do not affect mature DC.

In vivo immunization and tumor protection. To test the ability ofDef2-scFv38, Def3-scFv38, BP-1-scFv38, HNP-2-scFv38, HNP-3-scFv38,HBD1-scFv38, and HBD2-scFv38 to render self tumor antigen, sFv38,immunogenic when immunized as genetic vaccine in mice, ten mice pergroup are gene-gun immunized with plasmid encoding fusions with maturedefensin fusions HNP-1-scFv38, HNP-2-scFv38, HNP-3-scFv38, HBD1-scFv38,and HBD2-scFv38, in order to demonstrate whether these fusions cantarget APC in vitro. As a control, mice are immunized with the similarDNA constructs but encoding inactive pro-defensin fusions (designated,e.g., pproDef2sFv38). Murine defensins induce significant anti-idiotypespecific antibodies compared to the prototype Ig38-KLH protein vaccine.In contrast, no anti-idiotypic antibody response is seen afterimmunization with pro-Defensin fusions. Thus, non immunogenic sFv isrendered immunogenic by mature defensins, and the response correlateswith their ability to induce chemotaxis of immature DC and other APC.

Groups of ten mice immunized with defensin or control plasmids arechallenged with 20-fold lethal dose of tumor two weeks after the last ofthree serial immunizations. Control mice are immunized with inactivepro-defensin-sFv38 fusions or with corresponding active defensins fusedwith sFvA20 from irrelevant lymphoma A20 sFv or PBS. Mice immunized withinactive pproDef2sFv38 do not survive. In contrast, significantprotective immunity is elicited in mice immunized with pDef2sFv38 andpDef3sFv38. The survival closely correlates with the presence offunctionally intact or active defensins which can act on immature DC orother, APC via differentially expressing their receptors. AlthoughGen-gun bombarded DNA-would target variety of skin cells including skinAPC and LC, it is not sufficient to render self-tumor antigen (sFv)immunogenic because it required physical linkage of sFv with defensinmoieties. No positive humoral response or survival is detected in miceimmunized with DNA plasmids expressing free unlinked beta defensin orviral chemokine and sFv38.

Thus, the animal experiments clearly correlate with functional data fromin vitro studies. Id-specific responses and tumor immunity is detectedonly when the defensin moiety in the fusion retains the functionalproperties of the respective defensin, while no immunity is observedwhen the defensin is replaced with a mutant, functionally inactivedefensin. Furthermore, induction of chemotaxis to the site of vaccineinjection or production is not sufficient to generate immunity, asdemonstrated by the lack of humoral and anti-tumor immunity in miceimmunized with defensin alone or with either a mixture of free, unlinkeddefensin and sFv or defensin fused with an irrelevant sFv.

Example 2

Six- to nine-week old female C3H/HeN mice are immunizedintraperitoneally (i.p.) with 100 to 200 μg of the soluble protein inPBS and control immunogen Id38-KLH two times at two week intervals orare shaved and immunized by Accell gene delivery device (Agracetus,Inc., Middleton, WN) with 1μ gold particles carrying 1-3 μg plasmid DNA.Sera are collected by orbital bleeding two weeks after each vaccination.Serum anti-idiotypic (anti-Id) antibody levels are tested as described(111) over microtiter plates coated with 10 μg/ml native IgM 38c13. Twoweeks after the last immunization, mice are inoculated with 2000 38c13tumor cells i.p. Survival is determined, and significance with therespect to time to death, is assessed using BMDP IL software (BMDPstatistical software, Los Angeles). Mice are observed daily for anysigns of toxicity and date of death and animals surviving>80 days aftertumor challenge are killed and reported as long-term survivors.

Mice are immunized either with a plasmid coding for Def2-scFv38,Def3-scFv38, HNP-1-scFv38, HNP-2-scFv38, HNP-3-scFv38, HBD1-scFv38,HBD2-scFv38, or a mixture of DNA constructs expressing unlinked scFv38and vMIPIscFv20A (scFv38D+vMIPIscfv20AD).

Ten mice per group are immunized with two types of scFv38 fused to Def2,Def3, HNP-1, HNP-2, HNP-3, HBD1, or HBD2, differing only in orientationof variable genes in scFv. Control mice receive IgM-KLH (Id38-KLH) andthe corresponding defensin fusion to A20 lymphoma scFv (IP10scFv20A).Ten mice per group are immunized i.d. with plasmid coding either fordefensin fusion vaccine (Def2-scFv38, Def3-scFv38, HNP-1-scFv38,HNP-2-scFv38, HNP-3-scFv38, HBD1-scFv38, and HBD2-scFv38), or free scFv(scFv38D), or viral epitope preS2 fused scFv (PreS2scFv38D).

Effector CD8⁺ and CD4⁺ cells are depleted two weeks after the lastimmunization with three i.p. injections of 400 μg α-CD8 mAb 53.6.72, orα-CD4 mAb GK1.5 (both ammonium sulfate purified ascites, BiologicalResource Branch, NCI-FCRDC) (32, 34), or control rat IgG (Sigma).Control mice are immunized with plasmid expressing Def2, Def3, HNP-1,HNP-2, HNP-3, HBD1, or HBD2 fused to A20 scFv (MCP3scFv20AD).

Ten Balb/C mice per group are immunized i.p. twice with 100 μg of Def2,Def3, HNP-1, HNP-2, HNP-3, HBD1, or HBD2 fused with scFv20A protein inPBS and challenged i.p. with 10⁵ A20 tumor cells. To determine the roleof free versus linked defensin, defensin-scFv20A is co-injected with thesame defensin fused to an irrelevant scFv38(defensin-scFv20A+defensin-scFv38). Control mice are immunized with A20IgM-KLH (Id20A-KLH).

Immunoassays and serum anti-idiotypic antibody. The assessments forcorrect folding of purified scFv38 and fusion scFv38 are determined byELISA with mAbs and by inhibition assay with Id38-KLH sera (immunizedwith native IgM 38c13 conjugated to KLH). Briefly, microtiter plates(Nunc, Naperville, Ill.) are coated overnight at 4° C. with 10 μg/mlanti-c-myc mAb 9E10 in carbonate buffer (50 mM NaHCO₃, pH 9.0). Thewells are blocked with 5% nonfat dry milk in PBS for 30 min. Plates arewashed in 0.05% Triton X-100 in PBS, and serially diluted scFv (startingfrom 10 μg/ml in 50 μl 2% BSA/PBS) is applied, after which plates areincubated 40 min at room temperature. After washing, the wells areincubated with 50 μl of 1:300 diluted biotinylated anti-Id38 mAb in 2%BSA/PBS for 30 min at room temperature. Wells are washed and incubatedwith streptavidin-HRP conjugate (1:5000) in 2% BSA/PBS for 30 min atroom temperature. Then, wells are washed and incubated with ABTSperoxidase substrate (KPL, Gaithersburg, Md.) and the absorbance at 405nm is measured.

Inhibition assays are performed as described above, except plates arecoated with 10 μg/ml of native IgM 38c13, then wells are incubated for30 min at room temperature with a 1:2 dilution of positive Id38-KLH seramixed with serially diluted purified scFv proteins starting from 50μg/ml in 2% BSA/PBS. The bound antibodies from the sera are assayed byincubating wells for 30 min at room temperature with anti-mouse IgG-HRPmAb (Jackson).

Serum anti-idiotypic (anti-Id) antibody levels are tested as described(37). Briefly, mouse serum is serially diluted over microtiter platescoated with 10 μg/ml native IgM 38c13. Binding of antibodies in theserum to 38c13 IgM is detected by goat anti-mouse IgG-HRP. Serum anti-Idantibody levels are quantitated by comparing sera titration curves witha standard curve obtained with a known concentration of a mixture ofpurified monoclonal anti-Id antibodies. Antibody levels are expressed ing/ml of serum for individual mice. In each ELISA, sera obtained frommice immunized with control IgM-KLH are included as negative controls.Such sera never showed any titration binding activity on Id-38c13.

In vitro and in vivo chemotaxis assays. Single cell suspensions areprepared from spleens of untreated C3H/HeJ mice. Murine T cellenrichment columns (R&D System, Minneapolis, Minn.) are then used toprepare a purified murine T cell population via high-affinity negativeselection according to the manufacturer's instructions. The isolationprocedure typically yields over 89% CD3⁺ T cells, as determined by FACSanalysis. T cell migration in vitro is assessed by 48-wellmicrochemotaxis chamber technique. Briefly, a 26 μl aliquot of therecombinant scFv fusion protein serially diluted in the chemotaxismedium (RPMI 1640, 1% BSA, 25 mM HEPES) is placed in the lowercompartment and 50 μl of cell suspension (5×10⁶ cells/ml) is placed inthe upper compartment of the chamber. The two compartments are separatedby a polycarbonate filter (5 μm pore size; Neuroprobe, Cabin John, Md.)coated with 10 μg/ml of fibronectin (Sigma, St. Luis, Mo.) and incubatedovernight at 4° C. or for 2 hours at 37° C. The chemotaxis assay isperformed at 37° C. for 2 hours. Then the filter is removed, fixed andstained with Diff-Quik (Harlew, Gibbstown, N.J.). The number of migratedcells in three high power fields (400×) is counted by light microscopyafter coding the samples. The results are expressed as the mean±SE valueof the migration in triplicate samples.

Migration of splenocytes and bone marrow-derived immature dendriticcells in vitro is assessed by the 48-well micro chemotaxis chambertechnique as described (112).

In order to test in vivo effects on cell accumulation, C3H/HeN mice areinjected s.c. with a single 10 μg dose of scFv fusion proteins. Portionsof the skin from the site of injection are removed 72 hours after theinjection, fixed in 10% neutral buffered formalin, embedded in paraffin,sectioned at 5 μm and stained with hematoxylin and eosin (H&E). Slidesare evaluated microscopically without knowledge of the experimentaltreatment.

In vivo cellular infiltration into murine skin. The numbers of PMN andmononuclear (MN) cells infiltrated into murine skin are graded asfollowing:—, no significant lesion; 1, mild; 2, moderate; 3 severe; F,focal; MF, multi focal. Mice are injected with 10 μg of Def2-scFv38,Def3-scFv38, HNP-1-scFv38, HNP-2-scFv38, HNP-3-scFv38, HBD1-scFv38, andHBD2-scFv38, or PBS, subcutaneously. After 72 h, the injection site isexcised and examined histologically on coded slides to determine theextent of infiltration. The amount of endotoxin injected with samples is0.5-1 units.

The traditional approach to enhance immunogenicity by cross linking toKLH is not effective. Several different approaches are used for theproduction of single chain antibody fragments from 38c13 cells (scFv38)in E. coli. Yield of scFv38 differ significantly depending on the methodused. Production of scFv38 through a secretory path using a PelB leadersequence as a native protein is least efficient. The problem is solvedwhen scFv38 is produced as insoluble “inclusion” bodies, which yieldabout 2-8 mg of refolded scFv per liter of the batch culture withgreater than 90% purity. Folding properties of the produced scFv38 aremonitored by either (i) inhibition assay with native Id38; or (ii)modified ELISA assay where scFv38 is captured through an anti-c-myc tagand detected with the biotinylated monoclonal anti-Id38 antibody(anti-Id38 mAb does not recognize linear or incorrectly folded epitope).These experiments demonstrate that scFv38, but not irrelevant scFv20A,specifically binds to anti-Id38c mAb and inhibits binding of the nativeId38c to anti-Id38c mAb, 50% binding inhibition by 10-15 fold excess ofscFv38. In addition, positive sera from Id38c-KLH immunized micespecifically recognizes purified scFv38. These data indicate thatpurified scFv38 is folded correctly and imitates the idiotype of thenative antibody (Id38c) of B cell lymphoma 38c13.

Immunization experiments show that scFv38, similarly to the native Id38cIgM, is a poor immunogen. Attempts were made to convert scFv38 into apotent immunogen by chemical cross linking with KLH, in analogy to thenative Id38c. However, in contrast to Id38-KLH, i.p. immunizations ofsyngeneic mice with 100 μg of scFv38-KLH did not elicit any anti-Id38cspecific antibody response. This inability to induce anti-Id38 responsecorrelates with the loss of ability to affect binding of anti-Id38 mAb(SIC5) to Id38c by samples containing scFv38-KLH, while a control sampleof an equimolar mixture of non-cross linked scFv38 and KLH (scFv38+KLH)inhibited anti-Id38/Id38c binding similarly to pure scFv38. These dataindicate that a fragile Id conformation of scFv38 is removed by KLHcross linking and that this traditional approach is not applicable forthe enhancement of immunogenicity of scFv38.

Design and Production of Defensin Fused scFv38. Def2-scFv38,Def3-scFv38, HNP-1-scFv38, HNP-2-scFv38, HNP-3-scFv38, HBD1-scFv38, andHBD2-scFv38 are subcloned from the relevant cell lines by RT/PCR usingspecific primers as described herein and inserted in frame in front ofthe scFv38 DNA sequence. The resulting fusion genes are designated asDef2-scFv38, Def3-scFv38, HNP-1-scFv38, HNP-2-scFv38, HNP-3-scFv38,HBD1-scFv38, and HBD2-scFv38, respectively. In order to evaluate inputof the immunoglobulin V chain specific orientation, two variants offusion defensin-scFv genes are designed, one containing a V_(H)-V_(L)and one containing a V_(L)-V_(H) sequence, respectively designated asscFv38 MH and scFv38(INV)MH.

All fusion proteins used in these experiments are purified frominclusion bodies of E. coli, solubilized and refolded as describedherein. A spacer sequence, as described herein, is introduced into thedefensin fusion proteins and correct folding is tested for eachrecombinant protein.

The ability of Def2-scFv38, Def3-scFv38, HNP-1-scFv38, HNP-2-scFv38,HNP-3-scFv38, HBD1-scFv38, and HBD2-scFv38 proteins to induce chemotaxisin vivo in C3H/HeN mice is also tested. Mice are s.c. injected once with10 μg of the fusion protein and after 72 hours, the skin around the siteof injection is removed and analyzed as described herein.

Production of fusion polypeptides comprising a human defensin and ahuman tumor antigen or HIV antigen. To produce the fusion polypeptidesof the present invention which comprise a defensin region and a humantumor antigen region or HIV antigen region, the following procedures arecarried out: Tumor or viral antigen is cloned by PCR or RT/PCR from DNAor RNA of biopsy cells of a patient, using specific primer. The primersare made using standard methods for selecting and synthesizing primersequences from analysis of known sequences of the genes of interest(e.g., from GenBank, Kabat Ig sequence database and other availablegenetic databases, as are known in the art). For example, lymphoma ormyeloma-specific scFv is cloned by RT/PCR from the nucleic acid from apatient's lymphoma or myeloma biopsy cells or from nucleic acid fromhybridoma cells expressing the patient's immunoglobulin. Several sets ofprimers are used to clone human variable (V) genes based on GenBank andKabat IG sequence data. As in cloning murine scFv, human tumor Vfragments are cloned and sequenced using a family-specific primer orprimer mixture for leader and constant region sequences. Next, scFv isconstructed using primers based on the sequence of each V gene cloned.These primers can have specific restriction endonuclease sites tofacilitate routine cloning, or scFv is made by overlapping PCR,according to methods well known in the art. The vector expressing thefusion polypeptide can contain several unique restriction endonucleasesites (e.g., XhoI, BamHI) between the 3′ end of the spacer sequence andthe 5′ end of the c-myc and six His tag sequences, or the 5′ end of thepolyA transcription terminator region (if a SmaI site is used), thusenabling routine cloning of any scFv, tumor antigen or viral antigen.

As described herein, nucleic acid encoding the defensin-tumor antigenfusion polypeptides of this invention is expressed in yeast (e.g.,Saccharomyces cerevisiae; Pichia pastoris, etc.) or in mammalian cellculture according to methods standard in the art. The proteins producedin these systems are affinity purified with anti-c-myc antibodies (e.g.,9E10; M5546, Sigma) or anti-poly-His antibodies (e.g., H1029, Sigma).Alternatively, immobilized metal chelate affinity chromatography (Ni-NTAresin, Qiagen) is used for purification of soluble or refolded fusionpolypeptides.

Administration of fusion polypeptides to human subjects. Immunity andsuppression of tumor growth in a human subject. To elicit a tumor cellgrowth-inhibiting response in a human subject, a fusion polypeptidecomprising a defensin and a tumor antigen which is present in the humansubject is administered to the subject subcutaneously in a dose rangingfrom 1 to 500 μg of the fusion polypeptide once weekly for about eightweeks or once monthly for about six months. Within the first monthfollowing the initial immunization, blood samples can be taken from thesubject and analyzed to determine the effects of administration of thefusion polypeptide. Particularly, the presence in the subject's serum,of antibodies reactive with the tumor antigen in the fusion protein canbe determined by ELISA, Western blotting or radioimmunoprecipitation, orother methods for detecting the formation of antigen/antibody complexesas would be standard practice for one of ordinary skill in the art ofimmunology. Also, a cellular immune response to the tumor antigen in thefusion polypeptide can be detected by peripheral blood lymphocyte (PBL)proliferation assays, PBL cytotoxicity assays, cytokine measurements, orother methods for detecting delayed type hypersensitivity and cellularimmune response, as would be standard practice for one of ordinary skillin the art of immunology. Additionally, the kinetics of tumor growth andinhibition of tumor cell growth can be determined by monitoring thesubject's clinical response, through physical examination, tumormeasurement, x-ray analysis and biopsy. The exact dosage can bedetermined for a given subject by following the teachings as set forthherein, as would be standard practice for one of ordinary skill in theart of vaccine development.

Example 3

Using various chemokines and defensins, this example demonstrates thatprotective antitumor immunity can be obtained by targeting immature, butnot mature DC. Thus, MIP-3α and β-defensins render otherwisenon-immunogenic tumor antigens immunogenic and induce protective andtherapeutic antitumor immunity. In contrast, immunizations withhomeostatic chemokines SLC or SDF1β do not elicit antitumor immunity.While both humoral and cellular immune responses are useful fortreatment of the more aggressive 38C13 tumor that expresses IgMprimarily on its surface (Campbell, M. J., L. Esserman, N. E. Byars, A.C. Allison, and R. Levy. 1990. Idiotype vaccination against murine Bcell lymphoma. Humoral and cellular requirements for the full expressionof antitumor immunity. J. Immunol. 145:1029; Biragyn, A., Tani, K.,Grimm, M. C., Weeks, S. D., and Kwak, L. W. 1999. Genetic fusion ofchemokines to a self tumor antigen induces protective, T-cell dependentantitumor immunity. Nature Biotechnology 17: 253) cellular immunity isprotective for slower growing A20 lymphoma, which largely secretes itsidiotypic antigen. Thus, the breadth of the disclosed compositions andmethods as a generally useful for vaccines was also made apparent by itsability to elicit for the first time eradication of established A20lymphoma.

Materials and Methods

Fusion gene cloning and plasmid constructions. Cloning strategy forlymphoma specific V_(H) and V_(L) fragments from 38C13 (Bergmanm Y. andHaimovich, J. 1977. Characterization of a carcinogen-induced murine Blymphocyte cell line of C3H/eb origin. J. Immunol. 7: 413) and A20 (Kim,K. J., Kanellopoulos, Langevin C., Merwin, R. M., Sachs, D. H., andAsofsky, R. 1979. Establishment and characterization of BALB/c lymphomalines with B cell properties. J. Immunol. 122(2): 549) cells as sFv38and sFv20, respectively, with MCP-3 and IP-10 are described elsewhere(Biragyn, A., Tani, K., Grimm, M. C., Weeks, S. D., and Kwak, L. W.1999. Genetic fusion of chemokines to a self tumor antigen inducesprotective, T-cell dependent antitumor immunity. Nature Biotechnology17: 253). Genes for mature murine β-defensins were cloned from LPS (10ng/ml) treated BALB/c mouse skin in frame to the 5′-end of sFv by RT/PCRfrom total RNA using specific primers as described previously (Biragyn,A., Tani, K., Grimm, M. C., Weeks, S. D., and Kwak, L. W. 1999. Geneticfusion of chemokines to a self tumor antigen induces protective, T-celldependent antitumor immunity. Nature Biotechnology 17: 253). Thefollowing pairs of primers were used for β-defensin 2 (GeneBank #AJ011800) PRmDF2β-5′ (ACCATGGAACTTGACCACTGCCACACC; SEQ ID NO:16) andPRmDFβ-3′ (TGAATTCAAGATCTTTCATGTACTTGCAACAGGGGTTGTT; SEQ ID NO:17) andfor β-defensin 3 (GeneBank # AF092929) PRmDF3β-5′(ACCATGGAAAAAATCAACAATCAGTAAGTTGTTTGAGG; SEQ ID NO:18) and PRmDF3β-3′(CTCGAGCTAGAATTCTTTTCTCTTGCAGCATTTGAGGAAA; SEQ ID NO:61). β-pro-defensin2 gene was cloned for eukaryotic expression using PRproDF2β L-5′(AAAGCTTCCACCATGAGGACTCTCTGCTCT; SEQ ID NO:19) and PRmDF2β-3′, whichcontained native secretion signal sequence. SDF-1β (GeneBank # HSU16752)was cloned from 10 ng/ml LPS treated human monocytes using PRhSDF1β-5′(CTCTAGACACCATGAACGCCAAGGTCGTGGTCGTGCTG; SEQ ID NO:20) and PRhSDF1β-3′(TGAATTCCATCTTGAACCTCTTGTTTAAAGCTTT; SEQ ID NO:21). Murine MIP-3α(GeneBank # AJ222694) was cloned from mixture of thymus and kidney cDNAusing PRmMIP3α-5′ (ACCATGGCAAGCAACTACGACTGTTGCCTC; SEQ ID NO:22) andPRmMIP3α-3′ (ATAGAATTCCATCTTCTTGACTCTTAGGCTGA; SEQ ID NO:23). Murine SLC(GeneBank # U88322) was recloned from the plasmid (gift of Dr. Shakhov,SAIC-Fredeick) using PRmSLC-5′ (ACCATGGATGGAGGGGGACAGGACTGCT; SEQ IDNO:24) and PRmSLC-3′ (ATAGAATTCTCCTCTTGAGGGCTGTGTCTGT; SEQ ID NO:25).All constructs were verified by DNA dideoxy-sequencing method (Amersham,USA) and purified using plasmid purification kit (Qiagen, Valencia,Calif.).

Recombinant fusion proteins purified as inclusion bodies after 8 hoursof induction in Super-Broth (Digene Diagnostics, Inc., Beltsville, Md.)with 0.8 mM IPTG as described elsewhere (Biragyn, A., Tani, K., Grimm,M. C., Weeks, S. D., and Kwak, L. W. 1999. Genetic fusion of chemokinesto a self tumor antigen induces protective, T-cell dependent antitumorimmunity. Nature Biotechnology 17: 253), and refolded according toBuchner et al (Buchner, J., Pastan, I., and Brinkmann, U. 1992. A methodfor increasing the yield of properly folded recombinant fusion proteins:single-chain immunotoxins from renaturation of bacterial inclusionbodies. Anal. Biochem. 205(2): 263). The refolded fusion proteins werepurified by heparin-sepharose chromatography (Pharmacia Biotech,Uppsala, Sweden). The integrity and purity (greater than 90%) ofrecombinant proteins were tested by PAGE and by western blothybridization with 9E10 anti c-myc mAb (Sigma). Correct folding ofpurified sFv38 proteins were determined by the ability to bind toanti-idiotype mAb SIC5 in ELISA (Biragyn, A., Tani, K., Grimm, M. C.,Weeks, S. D., and Kwak, L. W. 1999. Genetic fusion of chemokines to aself tumor antigen induces protective, T-cell dependent antitumorimmunity. Nature Biotechnology 17: 253). Briefly, serially diluted sFvwere added to microtiter plates coated with 10 mg/ml anti c-myc mAb9E10. After washing, plates were incubated with a 1:300 dilutedbiotin-labeled S1C5, followed with streptavidin-HRP (1:5000, JacksonLab., Inc., Bar Harbor, Me.) and developed with ABTS peroxidasesubstrate (KPL, Gaithersburg, Md.). Proteins were biotinilated usingEZ-Link Sulfo-NHS-LC-Biotin following manufacturer's protocol (Pierce).

Isolation of murine bone marrow derived dendritic cells (Fields, R. C.,Osterholzer, J. J., Fuller, J. A., Thomas, E. K., Geraghty, P. J., andMule, J. J. 1998. Comparative analysis of murine dendritic cells derivedfrom spleen and bone marrow. J. Immunother. 21: 323). Briefly, bonemarrow was collected from tibias and femurs of 4 to 6 months old BALB/cmice. Erythrocytes were lysed with ACK lysis buffer (BioWhittaker,Walkersville, Md.). CD8⁺, CD4⁺, B220⁺ and I-A^(b) cells were depletedusing a mixture of mAbs and rabbit complement. The mAbs were TIB-146(anti-B220), TIB-150 (anti-CD8), TIB-207 (anti-CD4), TIB-229(anti-I-A^(b)) obtained from ATCC. Cells were cultured in DC medium(RPMI 1640 containing 5% heat inactivated fetal bovine serum, 1%penicillin, streptomycin, 1% L-glutamine and 5×10⁻⁵ 2-ME) containing 10ng/mL each of murine IL-4 and GM-CSF (Peprotech). Adherent cells wereharvested on day 4 and day 7 and used in subsequent experiments. DC werematured by TNFα (10 ng/ml, PharMingen), overnight in DC medium iDC atday four—five cultivation were in general CD11⁺ (69%), B7.2⁺ andI-A^(b+) (21%), B7.2⁻ and I-A^(b+) (18%), CD40⁺ (27%). Upon maturationthe DC were CD11c⁺ (87%), B7.2+ and I-A^(b+) (62%), B7.2⁻ and I-A^(b+)(3%), CD40⁺ (87%). The following mAb used in FACS: CD-11c-APC, MAC3-PE,Gr-1-FITC, B220-PE, Thy 1.2-FITC, I-A^(b)-FITC, B7.2-PE, CD40-PE(PharMingen).

In vitro chemotaxis assay: The migration of DC (50 ml, 10⁶ cells/ml) wasassessed using a 48-well microchemotaxis chamber (Neuro Probe, CabinJohn, Mass.) with a 5-mm polycarbonate filter (Osmonics, Livermore,Calif.) as described (Falk, W., Goodwin, R. H. Jr, and Leonard, E. J.1980. A 48-well micro chemotaxis assembly for rapid and accuratemeasurement of leukocyte migration. J. Immunol Methods 33(3): 239; Yang,D., Chen, Q., Stoll, S., Chen, X., Howard, O. M., and Oppenheim, J. J.2000. Differential regulation of responsiveness to fMLP and C5a upondendritic cell maturation: correlation with receptor expression. J.Immunol. 165.(5.):2694). Cells were incubated at 37° C. in humidifiedair with 5% CO₂ for 1.5 h. DC migrating across the filter were countedusing a Bioquant semiautomatic counting system. The results (as themean±SE of triplicate samples) are presented as chemotactic index (C.I.) defined as the fold increase in the number of migrating cells in thepresence of test factors over the spontaneous cell migration (in theabsence of test factors). Human MIP-3α and MIP-3β were from PeproTech(Rocky Hill, N.J.).

Cell lines and mice. The carcinogen-induced, C3H 38C-13 B cell lymphomais described by Bergmanm Y. and Haimovich, J. 1977. Characterization ofa carcinogen-induced murine B lymphocyte cell line of C3H/eb origin. J.Immunol. 7: 413. The 38C-13 tumor secretes and expresses IgM (k) on thecell surface. The BALB/c A20 lymphoma (Kim, K. J., Kanellopoulos,Langevin C., Merwin, R. M., Sachs, D. H., and Asofsky, R. 1979.Establishment and characterization of BALB/c lymphoma lines with B cellproperties. J. Immunol. 122(2): 549) was from the American Type CultureCollection (ATCC, Rockville, Md.) and expresses IgGk. Murine CCR6expressing HEK293 cells (HEK293/CCR6) were obtained from Dr. Farber, J.(NIAID/NIH).

Flow cytometric analysis. Trypsinized 2.5×10⁶ (HEK293/CCR6 or HEK293)cells/ml were incubated with 20 mg/ml biotinilated mMIP3asFv38 for 30min on ice in PBS with 2% BSA (PBS/BSA) and 20% mouse sera. Cells werestained on ice for 30 min with 0.2 mg/ml Streptavidin-PE (Pharmingen)and fixed with 1% paraformaldehyde.

In vivo immunizations and tumor protection experiment. Animal care wasprovided in accordance with the procedures outlined in a Guide for theCare and Use of Laboratory Animals (NIH Publication No. 86-23, 1985).Six- to nine-week old female C3H/HeNCrlBR or BALB/c mice (Charles RiverLaboratories, Frederick, Md.) were used. Mice (10 per group) wereimmunized with Helios Gene Gun System (Bio-Rad, Hercules, Calif.) with1-2 mg plasmid DNA three times every two weeks as described (Biragyn,A., Tani, K., Grimm, M. C., Weeks, S. D., and Kwak, L. W. 1999. Geneticfusion of chemokines to a self tumor antigen induces protective, T-celldependent antitumor immunity. Nature Biotechnology 17: 253.). Two weeksafter the last immunization, mice were challenged i.p. with 2×10³ or2.5×10⁵ 38C13 or A20 lymphoma cells, respectively, and followed forsurvival. Differences in survival between groups were determined bynon-parametric logrank test (BMDP statistical software, Los Angeles).P-values refer to comparison with group immunized with DNA expressingthe same chemokine or defensin fused with an irrelevant sFv, or sFvfused with mutant chemokine, unless specified.

Therapy of established tumor with DNA vaccine. Six- to nine-week oldfemale BALB/c mice (ten per group) were challenged with 2.5×10⁵syngeneic A20 tumor cells. At day 1, 4, 8 and 18 these mice weregene-gun immunized with DNA plasmid (containing about 1-2 mg DNA perimmunization) and mice followed for tumor progression.

Adoptive transfer experiments. BALB/c mice were gene gun immunized with1-2 mg pMCP3sFv20 twice biweekly and splenocytes and sera were removedten days after the last immunization. Ten BALB/c mice per group werei.p. injected in saline with 2.5×10⁵ A20 tumor cells per mouse mixedwith 2×10⁷ splenocytes or sera from immune or mock treated mice and micefollowed for tumor progression.

Results

Murine β-defensins and chemokines retain their functional integrity asfusion proteins with sFv and chemo-attract immature, but not mature, DC.First, a variety of chemokine and β-defensin fusion proteins were clonedand purified with sFv, a lymphoma Ig-derived non-immunogenic Fv from thetwo different B cell lymphomas 38C-13 and A20 (Table 1).

TABLE 1 Ligand-Antigen fusion constructs DNA vaccine Ligand: Defensin orname chemokine Antigen Protein Name Description Antigen alone psFv38none sFv38 sFv38 Single chain antibody fragment from 38C-13 lymphomapsFv20 none sFv20 sFv20 Single chain antibody fragment from A20 lymphomaDefensin fusions: pmDF2βsFv38 murine β-defensin 2 sFv38 mDF2βsFv38Murine β-defensin 2 fusion with sFv38 pmDF3βsFv38 murine β-defensin 3sFv38 mDF3βsFv38 Murine β-defensin 3 fusion with sFv38 pproDF2βsFv38murine pro-β-defensin 2 sFv38 mproDF2βsFv38 Murine pro-β-defensin 2fusion with sFv38 pmDF2βsFv20 murine β-defensin 2 sFv20 mDF2βsFv20Murine β-defensin 2 fusion with sFv20 pmSF3βsFv20 murine β-defensin 3sFv20 mDF3βsFv20 Murine β-defensin 3 fusion with sFv20 Inflammatorychemokine fusions: pmMIP3αsFv38 murine MIP3α sFv38 mMIP3αsFv38 MurineMIP3α fused with sFv38 pmMIP3αsFv20 murine MIP3α sFv20 mMIP3αsFv20Murine MIP3α fused with sFv20 pmIP10sFv38 murine IP-10 sFv38 mIP10sFv38Murine IP-10 fused with sFv38 pmIP10sFv20 murine IP-10 sFv20 mIP10sFv20Murine IP-10 fused with sFv20 pMCP3sFv38 human MCP-3 sFv38 MCP3sFv38human MCP-3 fused with sFv38 pMCP3sFv20 human MCP-3 sFv20 MCP3sFv20human MCP-3 fused with sFv20 Homeostatic chemokine fusions: pmSLCsFv38murine SLC sFv38 mSLCsFv38 murine SLC fused with sFv38 pmSDF1βsFv38human SDF1β sFv38 SDF1βsFv38 human SDF1β fused with sFv38 Controlfusions: pmDF3βMuc1 murine β-defensin 3 Muc1 mDF3βMuc1 Murine β-defensin2 fusion with Muc1 phMCP3-EGFP hMCP-3 EGFP hMCP3-EGFP human MCP-3 fusionwith EGFP phMDC-EGFP hMDC EGFP hMDC-EGFP human MDC fusion with EGFPpMC148MsFv38 viral, MC148 SFv38 MC148MsFv38 Viral MC148 fusion withsFv38 Protein vaccine: Ig38-KLH None Ig38 Ig38-KLH 38C-13 lymphomaderived IgM protein cross-linked with KLH Ig20-KLH None Ig20 Ig20-KLHA20 lymphoma derived IgG2a protein cross-linked with KLHFor example, sFv fusion proteins with murine inflammatory chemokineMIP-3α, murine β-defensin 2 and β-defensin 3 were designated mMIP3αsFv38, mDF2β sFv38 and mDF3β sFv38, or mMIP3α sFv20, mDF2β sFv20 andmDF3β sFv20, respectively. Similarly, sFv fusion proteins withhomeostatic chemokines SLC and SDF1β were designated mSLCsFv38 and SDF1βsFv38, respectively. Control proteins contained either sFv fusions withmutant chemokine, generated by replacing the first Cys residue with Seror by truncation of the amino-termini to abrogate a respective receptorbinding, or an inactive form pro-defensin 2, β-defensin 2 with itspro-sequence (mproDF2β sFv38, Table 1). All fusion proteins hadcomparable idiotype folding, as tested by inhibition ELISA withmonoclonal anti-Id antibodies, which bind only properly folded parentallymphoma Id.

The functional integrity of these proteins was tested by their abilityto induce chemotaxis of murine APC and THP-1 cells and binding to thechemokine receptor transfected cells. CCR6 transfected, but not parentalHEK293 cells specifically stained with biotinylated murine MIP3α fusionprotein. Furthermore, as expected, chemokine fusion proteins, but notcontrol mutant fusion proteins, induced chemotaxis of THP-1 cells ormurine DC. Since human β-defensin 2 was reported to interact with CCR6,murine β-defensin fusion proteins was tested for their ability to inducechemotaxis of murine CCR6 transfected cells, HEK293/CCR6. These cellswere chemo-attracted by murine MIP3α (a ligand of CCR6) fusion protein(mMIP3α sFv38). Both fusion proteins β-defensin 2 and 3, but not fusionproteins β-defensin 2 containing pro-sequence (mproDF2β 2sFv38) orcontrol viral chemokine (vMIP1MsFv38), chemo-attracted murine CCR6expressing cells, in a dose dependent manner. A control parental cellline, HEK293, which does not express CCR6 was not attracted to theseproteins. Next, we tested the ability of these proteins to attractmurine bone marrow derived DC (which are known to express CCR6;Sallusto, F., Palermo, B., Lenig, D., Miettinen, M., Matikainen, S.,Julkunen, I., Forster, R., Burgstahler, R., Lipp, M., and Lanzavecchia,A. 1999. Distinct patterns and kinetics of chemokine production regulatedendritic cell function. Eur. J. Immunol 29(5): 1617; Sallusto, F., P.Schaerli, P. Loetscher, C. Schaniel, D. Lenig, C. R. Mackay, S. Qin, andA. Lanzavecchia. 1998. Rapid and coordinated switch in chemokinereceptor expression during dendritic cell maturation. Eur. J. Immunol28:2760) was tested. The purity of murine bone marrow derived DC cellsand their immature phenotype at day four of cultivation was confirmed byexpressions of CD11c⁺ and low levels of I-A, B7.2 and CD40 respectively(see Methods). Similarly to MIP3α, both β-defensin fusion proteinsinduced chemotaxis of immature DC in a dose dependent manner with peakactivity at 10 ng/ml and 100 ng/ml for mDF2β sFv38 and mDF3bsFv38,respectively. The predominant immature phenotype of these DC was alsosupported by their ability to migrate to human MIP3α, a chemo-attractantspecific for CCR6⁺ immature DC (Dieu, M. C., Vanbervliet, B., Vicari,A., Bridon, J. M., Oldham, E., Ait-Yahia, S., Briere, F., Zlotnik, A.,Lebecque, S., and Caux, C. 1998. Selective recruitment of immature andmature dendritic cells by distinct chemokines expressed in differentanatomic sites. J. Exp. Med. 188(2): 373; Yang, D., Howard, O. M., Chen,Q., and Oppenheim, J. J. 1999. Cutting edge: immature dendritic cellsgenerated from monocytes in the presence of TGF-beta 1 expressfunctional C-C chemokine receptor 6. J. Immunol 163(4): 1737), and theirlimited ability to react to human MIP3β (Sallusto, F., Palermo, B.,Lenig, D., Miettinen, M., Matikainen, S., Julkunen, I., Forster, R.,Burgstahler, R., Lipp, M., and Lanzavecchia, A. 1999. Distinct patternsand kinetics of chemokine production regulate dendritic cell function.Eur. J. Immunol 29(5): 1617), a chemo-attractant specific for CCR7⁺mature DC. In contrast, none of the defensin and MIP3α fusion proteinsstimulated chemotaxis of TNF-induced mature DC, which migrated to MIP3β.Furthermore, control fusion protein with mutant MIP3a or mproDF2β sFv38did not induce chemotaxis of any DC. Therefore, murine β-defensin 2 and3 fusion proteins can specifically target iDC and induce theirCCR6-mediated chemotaxis, similarly to MIP3α.

Effect of Murine β-Defensin or in Inflammatory Chemokine FusionConstructs on Capacity of Non-Immunogenic Tumor Antigens to InduceHumoral Immunity.

Lymphoma idiotype alone is non-immunogenic in syngeneic mice. Similarly,DNA immunizations with lymphoma derived Fv or sFv alone, particularlyfrom 38C13 and A20 lymphomas, do not induce immunity in syngeneic mice(Biragyn, A., Tani, K., Grimm, M. C., Weeks, S. D., and Kwak, L. W.1999. Genetic fusion of chemokines to a self tumor antigen inducesprotective, T-cell dependent antitumor immunity. Nature Biotechnology17: 253). Therefore, a fusion construct of inflammatory peptides, suchas MIP3α or β-defensins, with these sFv antigens was used to demonstratethat it would induce specific immunity when administered as a DNAvaccine in mice. Ten mice per group were immunized by gene-gun withplasmids encoding fusion proteins consisting of mature forms ofβ-defensins (pmDF2β sFv38 and pmDF3β sFv38, respectively) or MIP3α(pmMIP3α sFv38). Control mice were immunized with DNA constructsencoding sFv fused with inactive pro-Defensin (pproDF2β sFv38), ormutated and inactive chemokines (pvMC148MsFv38). As will be discussedlater, no antibody was generated when mice were immunized with 2 mg DNAexpressing a mixture of plasmids containing unlinked sFv and murineβ-defensin (pmDF3β Muc1+sFv38) or chemokine. In contrast, mice immunizedwith plasmids encoding sFv fusion proteins with murine β-defensins,MIP3α or SLC produced idiotype-specific antibody levels comparable tothe levels induced by vaccination with tumor-derived intact Ig proteinconjugated to KLH. However, control mice immunized with an inactivepro-β-defensin (pproDef2β sFv38) or mutant chemokine sFv fusionconstructs (pvMC148MsFv38), or sFv38 alone did not produce any anti-Idantibody responses. Interestingly, the two types of β-defensins differedin their capacity to elicit antibody responses elicited, though bothproduced predominantly specific IgG1 antibodies, β-defensin 3 wassuperior to β-defensin 2 for induction of specific antibodies to everyantigen tested.

β-defensin fusion vaccines elicit protective and therapeutic antitumorimmunity. The vaccine protocol used to elicit protective antitumorimmunity was as follows: first, immunizing mice with 2 mg DNA constructsthree times with biweekly intervals, then, two weeks after the lastimmunization, challenging them with a 20-fold lethal dose of syngeneictumor. No survival was observed in control groups immunized with PBS orplasmids encoding sFv38 fused with inactive pro-β-defensin-2 (pproDef2βsFv38), with irrelevant chemokine plasmid vaccines pMDC-EGFP or withmutant constructs pMC148MsFv38. In contrast, significant protectiveimmunity was elicited in mice immunized with pmDF2β sFv38 or pmDF3βsFv38 (logrank P<0.001 and 0.004 as compared with pproDF2β sFv38 andpMC148MsFv38, respectively). The protection elicited with bothβ-defensin constructs was comparable to one induced by Ig-KLH proteinvaccine, a prototype vaccine which consists of lymphoma derived IgMcross-linked with KLH, being successfully tested in phase III clinicaltrial (Bendandi, M., Gocke, C. D., Kobrin, C. B., Benko, F. A., Sternas,L. A., Pennington, R., Watson, T. M., Reynolds, C. W., Gause, B. L.,Duffey, P. L., Jaffe, E. S., Creekmore, S. P., Longo, D. L., and Kwak,L. W. 1999. Complete molecular remissions induced by patient-specificvaccination plus granulocyte-monocyte colony-stimulating factor againstlymphoma. Nat. Med. 5(10): 1171). Similarly, DNA immunizations withMIP3a fusions elicited potent tumor protection (pmMIP3α sFv38, logrankP<0.0001 as compared with pMC148MsFv38). In contrast, none of miceimmunized with constructs encoding sFv fusion with homeostatic chemokinemurine SLC were protected (pmSLCsFv38, logrank p*<0.02 compared withpmMIP3α sFv38), despite the fact that this SLC fusion constructgenerated anti-Id specific antibodies comparable to pmMIP3α sFv38vaccinated group. Moreover, no immunity was detected in mice immunizedwith constructs expressing human SDF1β, which binds to murine CXCR4.Therefore, these data demonstrate that fusion constructs with defensins,which target immature DC, can render a non-immunogenic tumor antigen(sFv) immunogenic and elicit protective antitumor immunity, even for avery aggressive lymphoma, 38C13, which kills all control mice within 20days post challenge.

Next, the fusion constructs were used to demonstrate treatment of anestablished tumor. The A20 model (Kim, K. J., Kanellopoulos, LangevinC., Merwin, R. M., Sachs, D. H., and Asofsky, R. 1979. Establishment andcharacterization of BALB/c lymphoma lines with B cell properties. J.Immunol. 122(2): 549), which is relatively slower growing, was used todemonstrate the therapeutic potency of the approach. The therapeuticefficacy of the DNA constructs was assessed. Ten mice per group tumorbearing mice immunized with 2 mg DNA vaccine expressing β-defensin 2 and3 fusions with sFv20 (pmMIP3α sFv20, pmDF2β sFv20 and pmDF3β sFv20)starting one day after challenge with a lethal dose of A20 tumor,followed by three booster vaccinations. No survivors were observed intumor bearing mice randomized to control treatment with the sameβ-defensin, but fused with sFv derived from the 38C-13 lymphoma (pmDF2βsFv38), suggesting that non-specific effects of β-defensins were notsufficient for tumor eradication. In contrast, a significant number ofsurviving mice was observed in the pmDF2β sFv20 treatment group (logrankP<0.002 compared with pmDF2β sFv38). It is notable that in this model,although the β-defensin 3 fusion vaccine also induced a superiorspecific antibody production, the vaccine was not able to elicitantitumor immunity. A superior antibody production also was generated byvaccinating with A20 lymphoma derived Ig-KLH protein (Biragyn, A., Tani,K., Grimm, M. C., Weeks, S. D., and Kwak, L. W. 1999. Genetic fusion ofchemokines to a self tumor antigen induces protective, T-cell dependentantitumor immunity. Nature Biotechnology 17: 253), though it was notsufficient to elicit both protective (Biragyn, A., Tani, K., Grimm, M.C., Weeks, S. D., and Kwak, L. W. 1999. Genetic fusion of chemokines toa self tumor antigen induces protective, T-cell dependent antitumorimmunity. Nature Biotechnology 17: 253) and therapeutic immunity in A20model. Thus, these data suggest that induction of humoral immunity isnot sufficient to eradicate A20 B cell lymphoma, and that fusions withinflammatory chemokines MCP-3 and MIP3α or α-defensin 2 induced specificcellular antitumor responses.

Requirement for chemokine receptor targeting with fusion constructs.Next it was demonstrated that β-defensin should be physically linkedwith sFv by immunizing with mixture of unlinked β-defensin and sFv. Tenmice per group immunized with a mixture of separate plasmids encodingβ-defensin 3 (pmDF3β Muc1T) and sFv antigen (sFv38) failed to elicit aspecific humoral response, thus, demonstrating a requirement for sFv tobe physically linked to b-defensin. Furthermore, these mice challengedwith lethal dose of syngeneic 38C13 tumor exhibited no protection. Thesedata also suggest that chemokine receptor engagement with chemokine- ordefensin-sFv fusion is essential for the induction of immunity, and itwas not sufficient to simply attract APC, or induce inflammation at thesite of production of sFv antigen, but that direct APC targeting withantigen fused to β-defensin or chemokine was required, presumably viathe involvement of chemotactic receptors. To further demonstrate this,receptor-mediated immunity was inhibited by injection of the competingligand. Ten per group C3H mice were immunized with either pmDF3β sFv38alone or mixed with DNA encoding β-defensin fused with irrelevantantigen (pmDF3β Muc1T). Sera of mice immunized with a plasmid encodingsFv protein fused with murine β-defensin 3 by itself or in presence ofan irrelevant plasmid contained about 300 mg/ml idiotype-specificantibodies on average, which was two-three fold higher than the levelsinduced by vaccination with tumor-derived intact Ig protein conjugatedto KLH. However, much lower levels (5-15 mg/ml) of specific antibodieswere detected in sera of mice co-immunized with pmDF3β sFv38 mixedcompeting pmDF3β Muc1T. Two weeks after the last immunization, all micewere challenged with 20-fold lethal dose of syngeneic 38C13 tumor. Nosurvival was observed in control groups immunized with PBS or plasmidencoding β-defensin 3 fused with an irrelevant antigen (pmDF3β Muc1T).Similarly, no protection was detected in mice co-immunized with pmDF3βsFv38 and competing pmDF3β Muc1T (pmDF3β Muc1T/pmDF3β sFv38). Incontrast, 40% of mice immunized with pmDF3β sFv38 were protected(Logrank P<0.001 compared with pmDF3β Muc1T). Therefore, these datasupport the view that immunity to non-immunogenic tumor antigens fusedwith defensins or chemokines depended on their ability to engagechemokine receptor(s).

Discussion

This example demonstrates that a non-immunogenic tumor antigen, lymphomaidiotype (Stevenson, F. K., Zhu, D., King, C. A., Ashworth, L. J.,Kumar, S., and Hawkins, R. E. 1995. Idiotypic DNA vaccines againstB-cell lymphoma. Immunol. Rev. 145: 211), is rendered immunogenic whengenetically fused with defensins. This immunity is correlated with theability of murine β-defensin 2 and 3 to induce chemotaxis of murine bonemarrow derived immature, but not mature DC. Human β-defensin-2 binds toCCR6 preferentially expressed on iDC and resting memory T cells (Dieu,M. C., Vanbervliet, B., Vicari, A., Bridon, J. M., Oldham, E.,Ait-Yahia, S., Briere, F., Zlotnik, A., Lebecque, S., and Caux, C. 1998.Selective recruitment of immature and mature dendritic cells by distinctchemokines expressed in different anatomic sites. J. Exp. Med. 188(2):373; Yang, D., Howard, O. M., Chen, Q., and Oppenheim, J. J. 1999.Cutting edge: immature dendritic cells generated from monocytes in thepresence of TGF-beta 1 express functional C-C chemokine receptor 6. J.Immunol 163(4): 1737).

Example 4

Methods

Fusion gene cloning and plasmid constructions. gp120 gene was clonedfrom the plasmid DNA containing portion of HIV-1 (isolate 89.6) in framewith IP-10 secretion signal sequence (pgp120) using primersPRM89.6ENV-5′ (AAAGTCGACAAAGAAAAAACGTGGGTCACAATCT; SEQ ID NO:26) andPR89.6ENV-3′ (ATTCCCGGGTTATTTTTCTCTTTGCACTGTTCTTCTC; SEQ ID NO:27).Cloning strategy for lymphoma for chemokine genes has been reportedelsewhere (Biragyn, A., Tani, K., Grimm, M. C., Weeks, S. D., and Kwak,L. W. 1999. Genetic fusion of chemokines to a self tumor antigen inducesprotective, T-cell dependent antitumor immunity. Nature Biotechnology17: 253). Briefly, genes for mature human and murine chemokines, such asMCP-3 and IP-10, and defensins were cloned in frame to the 5′-end ofgp120 by RT/PCR from total RNA using specific primers as describedpreviously (Biragyn, A., Tani, K., Grimm, M. C., Weeks, S. D., and Kwak,L. W. 1999. Genetic fusion of chemokines to a self tumor antigen inducesprotective, T-cell dependent antitumor immunity. Nature Biotechnology17: 253). For example: genes for human MDC (GeneBank # HSU83171) wascloned from LPS 10 ng/ml treated human monocytes using respectivelypairs of primers PRhMDC-5′ (CTCTAGACACCATGGCTCGCCTACAGACTGCACT; SEQ IDNO:28) and PRhMDC-3′ (TGAATTCTTGGCTCAGCTTATTGAGAATCA; SEQ ID NO:29);Murine β-defensin 2 (GeneBank # AJ011800) and β-defensin 3 (GeneBank #AF092929) genes were cloned from LPS (10 ng) Balb/c mouse skin usingpairs of primers, respectively PRmDF2β-5′ (ACCATGGAACTTGACCACTGCCACACC;SEQ ID NO:30) and PRmDF2β-3′ (TGAATTCAAGATCTTTCATGTACTTGCAACAGGGGTTGTT;SEQ ID NO:31) and PRmDF3β-5′ (ACCATGGAAAAAATCAACAATCAGTAAGTTGTTTGAGG;SEQ ID NO:32) and PRmDF3β-3′ (CTCGAGCTAGAATTCTTTTCTCTTGCAGCATTTGAGGAAA;SEQ ID NO:33). Similarly, murine β-pro-defensin 2 gene was cloned foreukaryotic expression using PrproDF2βL-5′(AAAGCTTCCACCATGAGGACTCTCTGCTCT; SEQ ID NO:34) and PRmDF2β-3′, whichcontained native secretion signal sequence. gp120 was fused in framewith coding sequences from murine β-defensin 2 or 3, hMDC and hMCP3 togenerate DNA constructs pmDF2β gp120, pmDF3β gp120, phMDCgp120 andphMCP3gp120, respectively. Chemokine and defensins have been fused withgp120 through a spacer sequence NDAQAPKS (SEQ ID NO:35). To generateconstructs encoding mutant chemokines, the first Cys residue wasreplaced to Ser for all chemokines, except for hMCP-3 and hMDC where theamino-terminus up to the second Cys residue were truncated. Allconstructs were verified by DNA dideoxy-sequencing method, using T7sequenase kit (Amersham, USA) and purified using Qiagen plasmidpurification kit (Qiagen, Valencia, Calif.).

In vitro chemotaxis assay: The chemotactic migration of murine dendriticcells was assessed using a 48-well microchemotaxis chamber technique aspreviously described (Falk, W., Goodwin, R. H. Jr, and Leonard, E. J.1980. A 48-well micro chemotaxis assembly for rapid and accuratemeasurement of leukocyte migration. J. Immunol Methods 33(3): 239; Yang,D., Chen, Q., Stoll, S., Chen, X., Howard, O. M., and Oppenheim, J. J.2000. Differential regulation of responsiveness to fMLP and C5a upondendritic cell maturation: correlation with receptor expression. J.Immunol. 165.(5.):2694). Briefly, different concentrations of 26 mlchemotactic factors or aliquots of sFv fusion protein, serially dilutedin chemotaxis medium (RPMI 1640, 1% BSA, 25 mM HEPES), were placed inthe lower compartment of the chamber (Neuro Probe, Cabin John, Mass.),and 50 ml of dendritic cells (10⁶ cells/ml) were added to wells of theupper compartment. The lower and upper compartments were separated by a5-mm polycarbonate filter (Osmonics, Livermore, Calif.). Afterincubation at 37° C. in humidified air with 5% CO₂ for 1.5 h, thefilters were removed, scraped, and stained. Dendritic cells migratingacross the filter were counted with the use of a Bioquant semiautomaticcounting system. The results are presented as chemotactic index (C. I.)defined as the fold increase in the number of migrating cells in thepresence of test factors over the spontaneous cell migration (in theabsence of test factors). The results are expressed as the mean±SE oftriplicate samples. MIP-3α and MIP-3β were purchased from PeproTech(Rocky Hill, N.J.).

HIV-1 env antibody and CTL assays. Five BALB/c female mice per groupwere immunized with DNA plasmids four times using gene-gun. Two weeksafter the last immunization, HIV-1 89.6 env specific CTL was assessed inspleens and Peyer's patches as described elsewhere (Belyakov, I. M.,Derby, M. A., Ahlers, J. D., Kelsall, B. L., Earl, P., Moss, B.,Strober, W., and Berzofsky, J. A. 1998. Mucosal immunization with HIV-1peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes andprotective immunity in mice against intrarectal recombinant HIV-vacciniachallenge. Proc. Natl. Acad. Sci. U.S.A 95(4): 1709). Briefly, immunecells from spleen or Peyer's patch were cultured at 5×10⁶ per/milliliterin 24-well culture plates in complete T cell medium (CTM): RPMI 1640containing 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100U/ml), streptomycin (100 mg/ml), and 5×10⁻⁵ M 2-mercaptoethanol. Threedays later 10% concanavalin A supernatant was added as a source of IL-2(T-STIM, Collaborative Biomedical Products, Bedford, Mass.). Spleen orPeyer's patch cells were stimulated in vitro with P18-89.6A9 peptide(IGPGRAFYA; SEQ ID NO:36) (Belyakov, I. M., Wyatt, L. S., Ahlers, J. D.,Earl, P., Pendleton, C. D., Kelsall, B. L., Strober, W., Moss, B., andBerzofsky, J. A. 1998. Induction of a mucosal cytotoxic T-lymphocyteresponse by intrarectal immunization with a replication-deficientrecombinant vaccinia virus expressing human immunodeficiency virus 89.6envelope protein. J. Virol. 72(10): 8264) for a 7-day culture periodsbefore assay. Cytolytic activity of CTL lines was measured by a 4-hourassay with ⁵¹Cr-labeled P815 cell targets. For testing the peptidespecificity of CTL, ⁵¹Cr-labeled P815 targets were pulsed for 2 hourswith peptide at the beginning of the assay or left unpulsed as controls.The percent specific ⁵¹Cr release was calculated as 100× (experimentalrelease−spontaneous release)/(maximum release−spontaneous release).Maximum release was determined from supernatants of cells that werelysed by addition of 5% Triton-X 100. Spontaneous release was determinedfrom target cells incubated without added effector cells (Belyakov, I.M., Derby, M. A., Ahlers, J. D., Kelsall, B. L., Earl, P., Moss, B.,Strober, W., and Berzofsky, J. A. 1998. Mucosal immunization with HIV-1peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes andprotective immunity in mice against intrarectal recombinant HIV-vacciniachallenge. Proc. Natl. Acad. Sci. U.S.A 95(4): 1709).

Serum anti-env antibodies assessed by ELISA on 5 mg/ml gp120 proteinfrom isolate 89.6 produced in vaccinia virus coated 96-well plate. Thebound antibodies were detected by goat anti-mouse Ig-HRP mAb (Caltag)and developed with ABTS peroxidase substrate (KPL, Gaithersburg, Md.).

In vivo immunizations. Animal care was provided in accordance with theprocedures outlined in a Guide for the Care and Use of LaboratoryAnimals (NIH Publication No. 86-23, 1985). Six- to nine-week old femaleBALB/c mice (Charles River Laboratories, Frederick, Md.) were immunizedwith Helios Gene Gun System (Bio-Rad, Hercules, Calif.) with plasmid DNAfour times every two weeks. The abdominal area of mice was shaved, and 1m gold particles (Bio-Rad, Hercules, Calif.) carrying 1-3 mg DNA wereinjected at 400 psi.

Results and Discussion.

Mammalian expression plasmid vectors were constructed by cloning gp120alone (pgp120), or as fusion constructs of gp120 with β-defensin 2 or 3,human MDC or MCP-3 (pmDF2β gp120, pmDF3β gp120, pMDCgp120 andpMCP3gp120, respectively, Table 2). All constructs expressed equivalentamounts of gp120 when transfected transiently in 293 cells. Purifiedfusion proteins with proinflammatory chemokines or murine β-defensinsgenerally retained chemokine functional integrity, such aschemo-attraction of murine bone-marrow derived immature, but not matureDC (see Example 3).

TABLE 2 Ligand-Antigen fusion constructs Ligand: DNA vaccine Defensin orname chemokine Antigen Description Antigen alone pgp120 none gp120 HIV-1gp120, 89.6. Defensin fusion: pmDF2βgp120 murine β- gp120 Murineβ-defensin defensin 2 2 fusion with gp120 Chemokine fusions: pMCP3gp120hMCP-3 gp120 human MCP-3 fusion with gp120 pMDCgp120 hMDC gp120 humanMDC fusion with gp120

BALB/c mice were immunized four times biweekly using a gene gun with DNAplasmids encoding gp120 alone, or fusion constructs of gp120 withβ-Defensin 2 or 3, human MDC or MCP-3 (pgp120, pmDF2β gp120, pmDF3βgp120, pMDCgp120 and pMCP3gp120, respectively). Two weeks after the lastimmunization, sera from these mice were tested for α-gp120 antibodiesusing recombinant gp120 produced in Vaccinia. As expected, control miceimmunized with DNA encoding gp120 alone did not induce any anti-gp120antibody (Putkonen, P., Quesada-Rolander, M., Leandersson, A. C.,Schwartz, S., Thorstensson, R., Okuda, K., Wahren, B., and Hinkula, J.1998. Immune responses but no protection against SHIV by gene-gundelivery of HIV-1 DNA followed by recombinant subunit protein boosts.Virology 250(2): 293). In contrast, mice immunized with pmDF2β gp120,pmDF3β gp120, phMDCgp120 and phMCP3gp120 fusions all produced hightiters of env-specific antibodies. These data suggest that the inabilityof gp120 to elicit humoral immunity was circumvented by fusion to eitherproinflammatory chemokines or β-defensins.

These DNA fusions were used to induce anti-gp120 cellular responses.Spleen cells from mice immunized with β-Defensin 2 or MCP-3 fusionconstructs demonstrated significant lysis of P815 target cells pulsedwith HIV-1 89.6 A9 peptide (pmDF2β gp120 and phMCP3g120, respectively,pulsed (*) vs. unpulsed targets). In contrast, no CTL were detected inmice immunized with pgp120 alone or PBS. These data thus demonstratethat immunizations with DNA plasmids encoding gp120 fused withproinflammatory chemokines or β-defensins induced significant gp120V3-loop specific systemic CTL. Mice immunized with DNA encoding gp120fused with MCP3- or β-defensin 2 elicited significant CTL activity inPeyer's patches, suggesting that these vaccines also induced mucosalimmunity. This data indicate that DNA immunizations in the skin caninduce mucosal immunity in addition to systemic immunity, a feature thathad not been demonstrated prior to the disclosed compositions.

In addition to targeting gp120 to professional APC, expressed fusionproteins may induce expression of co-stimulatory molecules andproduction of pro-inflammatory cytokines by various subsets of immatureDC in vivo. Moreover, Th1 or Th2 cells could be differentially attractedby chemokines, thus modulating immunity. For example, MCP-1 stimulatesIL-4 production (Karpus, W. J., N. W. Lukacs, K. J. Kennedy, W. S.Smith, S. D. Hurst, and T. A. Barrett. 1997. Differential CCchemokine-induced enhancement of T helper cell cytokine production. J.Immunol 158:4129) and thereby induces control Th2 polarization (Gu, L.,S. Tseng, R. M. Homer, C. Tam, M. Loda, and B. J. Rollins. 2000. Controlof TH2 polarization by the chemokine monocyte chemoattractant protein-1.Nature 404:407). Moreover, MDC has been reported to selectivelychemo-attract Th2 cells towards APC (Imai, T., M. Nagira, S. Takagi, M.Kakizaki, M. Nishimura, J. Wang, P. W. Gray, K. Matsushima, and O.Yoshie. 1999. Selective recruitment of CCR4-bearing Th2 cells towardantigen-presenting cells by the CC chemokines thymus andactivation-regulated chemokine and macrophage-derived chemokine. Int.Immunol 11:81). Comparison of MDC fusion vaccines to fusions with MCP-3,which binds multiple receptors, such as CCR1-4 expressed on variety ofAPC, demonstrated that each vaccine construct induced CL-gp120 antibodyproduction. However, the highest yield was observed in sera of miceimmunized with MDC fusions, which unlike to the MCP-3 or β-defensin 2fusion constructs, failed to induce α-gp120 CTL. In concordance,non-immunogenic tumor antigens fused with MDC did not induce therapeuticantitumor immunity in murine tumor models, despite production of highlevels of specific antibody. In contrast, tumor antigen fusionconstructs with both MCP-3 and β-defensin 2 induced moderate antibodyproduction and significant therapeutic antitumor immunity (see Example3). Therefore, these data show that preferential immunity may beelicited by differential use of chemokines or defensin. Thus, thedisclosed compositions and methods represent a very efficient and simpleapproach to generate systemic and mucosal immunity. These results alsostand in contrast to the widely accepted view that DNA vaccinationsalone cannot induce anti-gp120 HIV-1 responses without additionalprotein or recombinant viral vaccine boosts (Hanke, T., Samuel, R. V.,Blanchard, T. J., Neumann, V. C., Allen, T. M., Boyson, J. E., Sharpe,S. A., Cook, N., Smith, G. L., Watkins, D. I., Cranage, M. P., andMcMichael, A. J. 1999. Effective induction of simian immunodeficiencyvirus-specific cytotoxic T lymphocytes in macaques by using amultiepitope gene and DNA prime-modified vaccinia virus Ankara boostvaccination regimen. J. Virol. 73(9): 7524; Putkonen, P.,Quesada-Rolander, M., Leandersson, A. C., Schwartz, S., Thorstensson,R., Okuda, K., Wahren, B., and Hinkula, J. 1998. Immune responses but noprotection against SHIV by gene-gun delivery of HIV-1 DNA followed byrecombinant subunit protein boosts. Virology 250(2): 293).

Example 5

This example demonstrates that antigens elicit effective immunity whenthey are targeted to APC, particularly iDC, via chemokine receptors asfusions with proinflammatory chemokines factors. Moreover, this exampledemonstrates that proinflammatory factors such as murine β-defensinsinduce chemotaxis of immature, but not mature, DC and, thus, can serveeffectively as an carrier for targeting antigens to APC. Non-immunogenictumor antigens or xenogeneic HIV gp120 antigen were rendered effectivelyimmunogenic when immunized as fusion with murine β-defensin 2. Thisexample also demonstrates the use of xenogeneic human and viralchemokines such as Kaposi's sarcoma virus derived chemokine analogues ofhuman MIP-1s, designated vMIP1 and vMIP2 (Nicholas, J., V. R. Ruvolo, W.H. Burns, G. Sandford, X. Wan, D. Ciufo, S. B. Hendrickson, H. G. Guo,G. S. Hayward, and M. S. Reitz. 1997. Kaposi's sarcoma-associated humanherpesvirus-8 encodes homologues of macrophage inflammatory protein-1and interleukin-6. Nat. Med. 3:(3)287-292), which bind multiplereceptors on murine cells and which might circumvent the potentialdevelopment of autoimmunity against host chemokine carriers (Luster, A.D. 1998. Chemokines—chemotactic cytokines that mediate inflammation. N.Engl. J. Med. 338:(7)436-445; Pelchen-Matthews, A., N. Signoret, P. J.Klasse, A. Fraile-Ramos, and M. Marsh. 1999. Chemokine receptortrafficking and viral replication. Immunol Rev. 168:33-49:33-49). Inaddition, using a pair of viral chemokines that bind to CCR8, theagonist vMIP1 (Endres, M. J., C. G. Garlisi, H. Xiao, L. Shan, and J. A.Hedrick. 1999. The Kaposi's sarcoma-related herpesvirus (KSHV)-encodedchemokine vMIP-I is a specific agonist for the CC chemokine receptor(CCR)8. J. Exp. Med. 189:(12)1993-1998) and antagonist MC148, expressedby Molluscum contagiosum virus (MCV) (Luttichau, H. R., J. Stine, T. P.Boesen, A. H. Johnsen, D. Chantry, J. Gerstoft, and T. W. Schwartz.2000. A highly selective CC chemokine receptor (CCR)8 antagonist encodedby the poxvirus molluscum contagiosum. J. Exp. Med. 191:(1)171-180), wedemonstrate that antigen targeting alone in the absence of chemotaxis issufficient for induction of immunity.

Methods

Fusion gene cloning and plasmid constructions. Cloning strategy forlymphoma specific V_(H) and V_(L) fragments from 38C13 (Bergmanm Y. andJ. Haimovich. 1977. Characterization of a carcinogen-induced murine Blymphocyte cell line of C3H/eb origin. J. Immunol. 7:413417) and A20(Kim, K. J., L. C. Kanellopoulos, R. M. Merwin, D. H. Sachs, and R.Asofsky. 1979. Establishment and characterization of BALB/c lymphomalines with B cell properties. J. Immunol. 122:(2)549-554) cells as sFv38and sFv20, respectively, has been reported elsewhere (Biragyn, A., K.Tani, M. C. Grimm, S. D. Weeks, and L. W. Kwak. 1999. Genetic fusion ofchemokines to a self tumor antigen induces protective, T-cell dependentantitumor immunity. Nature Biotechnology 17:253-258). Genes for maturehuman and murine chemokines and defensins were cloned in frame to the5′-end of sFv by RT/PCR from total RNA using specific primers asdescribed previously (Biragyn, A., K. Tani, M. C. Grimm, S. D. Weeks,and L. W. Kwak. 1999. Genetic fusion of chemokines to a self tumorantigen induces protective, T-cell dependent antitumor immunity. NatureBiotechnology 17:253-258). For example: genes for human MDC (GeneBank #HSU83171) and SDF-1β (GeneBank # HSU16752) were cloned from LPS 10 ng/mltreated human monocytes using respectively pairs of primers PRhMDC-5′(CTCTAGACACCATGGCTCGCCTACAGACTGCACT; SEQ ID NO:37) and PRhMDC-3′(TGAATTCTTGGCTCAGCTTATTGAGAATCA; SEQ ID NO:38); and PRhSDF1β-5′(CTCTAGACACCATGAACGCCAAGGTCGTGGTCGTGCTG; SEQ ID NO:39) and PRhSDF1-3′(TGAATTCCATCTTGAACCTCTTGTTTAAAGCTTT; SEQ ID NO:40). Murine β-defensin 2(GeneBank # AJ011800) and β-defensin 3 (GeneBank # AF092929) genes werecloned from LPS (10 ng) Balb/c mouse skin using pairs of primers,respectively PRmDF2β-5′ (ACCATGGAACTTGACCACTGCCACACC; SEQ ID NO:41) andPRmDF2β-3′ (TGAATTCAAGATCTTTCATGTACTTGCAACAGGGGTTGTT; SEQ ID NO:42) andPRmDF3β-5′ (ACCATGGAAAAAATCAACAATCAGTAAGTTGTTTGAGG; SEQ ID NO:43) andPRmDF3β-3′ (CTCGAGCTAGAATTCTTTTCTCTTGCAGCATTTGAGGAAA; SEQ ID NO:44).Similarly, murine β-pro-defensin 2 gene was cloned for eukaryoticexpression using PrproDF2 μL-5′ (AAAGCTTCCACCATGAGGACTCTCTGCTCT; SEQ IDNO:45) and PRmDF2β-3′, which contained native secretion signal sequence.For bacterial expression, the signal sequence was removed usingPRmDF2β-5′ (ACCATGGCTGTTGGAAGTTTAAAAAGTATTGGA; SEQ ID NO:46) andPRmDF2β-3′. Viral chemokine genes vMIP-I (GeneBank # KSU74585) andvMIP-II (GeneBank # KSU67775) were cloned from BCBL-1 lymphoma cell lineinfected with HHV-8 (NIH AIDS Research & Ref. Reag. Program), usingpairs of primers, respectively PRvMIP1L-5′(TAAGCTTCCACCATGGCCCCCCGTCCACGTTTATGCT; SEQ ID NO:47) and PRvMIP1-3′(TGAATTCAGCTATGGCAGGCAGCCGCTGCATCAGCTGCCT; SEQ ID NO:48) andPRvMIP1IL-5′ (TAAGCTTCACCATGGACACCAAGGGCATCCTGCTCGT; SEQ ID NO:49) andPRvMIP1I-3′ (TGAATTCGCGAGCAGTGACTGGTAATTGCTGCAT; SEQ ID NO:50). Maturesequences of vMIP-I and vMIP-II for expression in bacterial system werecloned using the following pairs of primers: PRvMIP1M-5′(ACCATGGCGGGGTCACTCGTGTCGTACA; SEQ ID NO:51) and PRvMIP1-3′ andPRvMIP2M-5′ (ACCATGGGAGCGTCCTGGCATAGA; SEQ ID NO:52) and PRvMIP1I-3′.MC148 chemokine gene (GeneBank # U96749) was cloned from plasmid DNAcontaining a portion of Moluscum contagiosum virus type 1 genome (Damon,I., P. M. Murphy, and B. Moss. 1998. Broad spectrum chemokineantagonistic activity of a human poxvirus chemokine homolog. Proc. Natl.Acad. Sci. U.S.A 95:(11)6403-6407) using pairs of primers PRMC148L-5′(AAAGCTAGCACCATGAGGGGCGGAGACGTCTTC; SEQ ID NO:53) and PRMC148-3′(AGAATTCCAGAGACTCGCACCCGGACCATAT; SEQ ID NO:54) and PRMC148M-5′(ACCATGGCACTCGCGAGACGGAAATGTTGTTTGAAT; SEQ ID NO:55) and PRMC148-3′,respectively for eukaryotic and bacterial expression. Thecarboxy-terminus of sFv was fused in frame with a tag sequence codingc-myc peptide AEEQKLISEEDLA (SEQ ID NO:56) and six His, respectively.Chemokine and defensins have been fused with sFv through a spacersequence NDAQAPKS (SEQ ID NO:57). To generate constructs encoding mutantchemokines, the first Cys residue was replaced to Ser for allchemokines, except for hMCP-3 and hMDC where the amino-terminus up tothe second Cys residue were truncated. Bacterial expression vectorscontained only genes encoding mature peptide genes, while constructs forDNA vaccination were fused in frame to a leader sequence of IP-10 inpCMVE/AB, except for constructs designed for pMDCsFv38 and pSDF1βsFv38plasmids, which contained their native signal sequences. All constructswere verified by DNA dideoxy-sequencing method, using T7 sequenase kit(Amersham, USA) and purified using Qiagen plasmid purification kit(Qiagen, Valencia, Calif.).

For the second model, gp120 gene was cloned from the plasmid DNAcontaining portion of HIV-1 (isolate 89.6) in frame with IP-10 secretionsignal sequence (pgp120) using primers PRM89.6ENV-5′(AAAGTCGACAAAGAAAAAACGTGG GTCACAATCT; SEQ ID NO:58) and PR89.6ENV-3′(ATTCCCGGGTTATTTTTCTCTTTGCACTGTTCTTCTC; SEQ ID NO:59). Similarly, gp120was fused in frame with coding sequences from murine β-defensin 2, hMDCand hMCP3 to generate DNA constructs pmDF2βgp120, phMDCgp120,phMCP3gp120, respectively.

Recombinant fusion proteins purified as inclusion bodies after 8 hoursof induction in Super-Broth (Digene Diagnostics, Inc., Beltsville, Md.)with 0.8 mM IPTG in the presence of 150 μg/ml carbenicillin and 50 μg/mlampicillin at 30° C., and refolded according to Buchner, et al (Buchner,J., I. Pastan, and U. Brinkmann. 1992. A method for increasing the yieldof properly folded recombinant fusion proteins: single-chainimmunotoxins from renaturation of bacterial inclusion bodies. Anal.Biochem. 205:(2)263-270) with modifications (Biragyn, A., K. Tani, M. C.Grimm, S. D. Weeks, and L. W. Kwak. 1999. Genetic fusion of chemokinesto a self tumor antigen induces protective, T-cell dependent antitumorimmunity. Nature Biotechnology 17:253-258) from BL21(DE3) cells(Invitrogen, Milford, Mass.). The refolded fusion proteins were purifiedby heparin-sepharose chromatography (Pharmacia Biotech, Uppsala,Sweden). The integrity and purity of recombinant proteins were tested byPAGE under reducing conditions and by western blot hybridization with9E10 anti-c-myc mAb (Sigma). Purification usually yielded solubleprotein with greater than 90% purity. Correct folding of purified sFv38proteins were determined by the ability to bind to anti-idiotype mAbS1C5 by ELISA (Biragyn, A., K. Tani, M. C. Grimm, S. D. Weeks, and L. W.Kwak. 1999. Genetic fusion of chemokines to a self tumor antigen inducesprotective, T-cell dependent antitumor immunity. Nature Biotechnology17:253-258). Briefly, serially diluted sFv were added to microtiterplates coated with 10 μg/ml anti-c-myc mAb 9E10. After washing, plateswere incubated with a 1:300 diluting of biotinylated S1C5. Plates werewashed, incubated with streptavidin-HRP (1:5000, Jackson Immuno researchLab., Inc., Bar harbor, ME) and developed with ABTS peroxidase substrate(KPL, Gaithersburg, Md.).

Isolation of murine bone marrow derived dendritic cells. Murine bonemarrow derived DC were isolated as described elsewhere (Fields, R. C.,J. J. Osterholzer, J. A. Fuller, E. K. Thomas, P. J. Geraghty, and J. J.Mule. 1998. Comparative analysis of murine dendritic cells derived fromspleen and bone marrow. J. Immunother. 21:(5)323-339). Briefly, bonemarrow was collected from tibias and femurs of 4 to 6 months old BALB/cmice by flushing with PBS using a 10 mL syringe with a 27 gauge needle.Erytrocytes were lysed by treatment with ACK lysis buffer (BioWhittaker,Walkersville, Md.). Cells expressing CD8, CD4, B220 and I-A^(b) weredepleted using a mixture of mAbs and rabbit complement. The mAbs wereTIB-146 (anti-B220), TIB-150 (anti-CD8), TIB-207 (anti-CD4), TIB-229(anti-I-A^(b)) obtained from ATCC. Cells were then resuspended with DCmedium (RPMI 1640 containing 5% heat inactivated fetal bovine serum, 1%penicillin streptomycin, 1% L-glutamine and 5×10⁻⁵ 2-ME) supplementedwith 10 ng/mL recombinant murine IL-4 and 10 ng/mL recombinant murineGM-CSF (Peprotech) and cultured in 6 well plates (7×10⁵ cells/mL, 5mL/well). On day two, 200 μl DC medium containing 10 ng/mL of both IL-4and GM-CSF were added to each well. Then, at day four, 5 μl of DC mediumcontaining cytokines were added to each well after non-adherant cellswere removed, and cells were cultured for additional 4 days. Cells wereharvested on day 4 and day 7 and used in subsequent experiments.

In vitro chemotaxis assay: The chemotactic migration of murine dendriticcells was assessed using a 48-well microchemotaxis chamber technique aspreviously described (Falk, W., R. H. J. Goodwin, and E. J. Leonard.1980. A 48-well micro chemotaxis assembly for rapid and accuratemeasurement of leukocyte migration. J. Immunol Methods 33:(3)239-247;Yang, D., Q. Chen, S. Stoll, X. Chen, O. M. Howard, and J. J. Oppenheim.2000. Differential regulation of responsiveness to fMLP and C5a upondendritic cell maturation: correlation with receptor expression. J.Immunol. 165:(5)2694-2702). Briefly, different concentrations of 26 mlchemotactic factors or aliquots of sFv fusion protein, serially dilutedin chemotaxis medium (RPMI 1640, 1% BSA, 25 mM HEPES), were placed inthe lower compartment of the chamber (Neuro Probe, Cabin John, Mass.),and 50 μl of dendritic cells (10⁶ cells/ml) were added to wells of theupper compartment. The lower and upper compartments were separated by a5-μm polycarbonate filter (Osmonics, Livermore, Calif.). Afterincubation at 37° C. in humidified air with 5% CO₂ for 1.5 h, thefilters were removed, scraped, and stained. Dendritic cells migratingacross the filter were counted with the use of a Bioquant semiautomaticcounting system. The results are presented as chemotactic index (C. I.)defined as the fold increase in the number of migrating cells in thepresence of test factors over the spontaneous cell migration (in theabsence of test factors). The results are expressed as the mean±SE oftriplicate samples. MIP-3α and MIP-3β were purchased from PeproTech(Rocky Hill, N.J.).

Chemokine receptor binding assay. Binding assays were performed by usinga single concentration of radio-labeled MIP-1β or SDF-1α: (human[¹²⁵-I]-[Leu³, Gly⁴⁷]-MIP-1β and human [¹²⁵I]-SDF-1α, 2200 Ci/mmol, NENLife Science Products Inc., Boston, Mass.) in the presence of increasingconcentrations of unlabeled ligands (MIP-1β and SDF-1α obtained fromPeproTech, Rocky Hill, N.J.). Human HEK293 cells transfected with CCR5at 1×10⁶/sample were suspended in 200 μl binding medium composed ofRPMI1640, 1 mg/ml BSA, 25 mM HEPES, and 0.05% sodium azide, andincubated in duplicates at room temperature for 40 min. Afterincubation, the cells were pelleted through a 10% sucrose/PBS cushionand the radioactivity associated with cell pellets was determined in aγ-counter (Clinigamma-Pharmacia, Gaithersburg, Md.). The binding datawere then analyzed with a Macintosh computer program LIGAND (P. Munson,Division of Computer Research and Technology, NIH, Bethesda, Md.). Thedegree of competition for binding by unlabeled chemokines was calculatedas follows: % competition for binding=1−(cpm obtained in the presence ofunlabeled ligand/cpm obtained in the absence of unlabeled ligand)×100%.

HIV-1 env antibody and CTL assays. Five BALB/c female mice per groupwere gene-gun immunized with DNA plasmids four times using gene-gun. Twoweeks after the last immunization, HIV-1 89.6 env specific CTL wasassessed in spleens and Peyer's patches as described elsewhere(Belyakov, I. M., M. A. Derby, J. D. Ahlers, B. L. Kelsall, P. Earl, B.Moss, W. Strober, and J. A. Berzofsky. 1998. Mucosal immunization withHIV-1 peptide vaccine induces mucosal and systemic cytotoxic Tlymphocytes and protective immunity in mice against intrarectalrecombinant HIV-vaccinia challenge. Proc. Natl. Acad. Sci. U.S.A95:(4)1709-1714). Briefly, immune cells from spleen or Peyer's patchwere cultured at 5×10⁶ per/milliliter in 24-well culture plates incomplete T cell medium (CTM): RPMI 1640 containing 10% fetal bovineserum, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100mg/ml), and 5×10⁻⁵ M 2-mercaptoethanol. Three days later 10%concanavalin A supernatant was added as a source of IL-2 (T-STIM,Collaborative Biomedical Products, Bedford, Mass.). Spleen or Peyer'spatch cells were stimulated in vitro with P18-89.6A9 peptide (IGPGRAFYA;SEQ ID NO:60) (Belyakov, I. M., L. S. Wyatt, J. D. Ahlers, P. Earl, C.D. Pendleton, B. L. Kelsall, W. Strober, B. Moss, and J. A. Berzofsky.1998. Induction of a mucosal cytotoxic T-lymphocyte response byintrarectal immunization with a replication-deficient recombinantvaccinia virus expressing human immunodeficiency virus 89.6 envelopeprotein. J. Virol. 72:(10)8264-8272) for a 7-day culture periods beforeassay. Cytolytic activity of CTL lines was measured by a 4-hour assaywith ⁵¹Cr-labeled P815 cell targets. For testing the peptide specificityof CTL, ⁵¹Cr-labeled P815 targets were pulsed for 2 hours with peptideat the beginning of the assay or left unpulsed as controls. The percentspecific ⁵¹Cr release was calculated as 100× (experimentalrelease−spontaneous release)/(maximum release−spontaneous release).Maximum release was determined from supernatants of cells that werelysed by addition of 5% Triton-X 100. Spontaneous release was determinedfrom target cells incubated without added effector cells (Belyakov, I.M., M. A. Derby, J. D. Ahlers, B. L. Kelsall, P. Earl, B. Moss, W.Strober, and J. A. Berzofsky. 1998. Mucosal immunization with HIV-1peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes andprotective immunity in mice against intrarectal recombinant HIV-vacciniachallenge. Proc. Natl. Acad. Sci. U.S.A 95:(4)1709-1714).

Serum anti-env antibodies assessed by ELISA on 5 μg/ml gp120 proteinfrom isolate 89.6 produced in vaccinia virus coated 96-well plate. Thebound antibodies were detected by goat anti-mouse Ig-HRP mAb (Caltag)and developed with ABTS peroxidase substrate (KPL, Gaithersburg, Md.).

Tumor cell lines and mice. The carcinogen-induced, C3H 38C-13 B celllymphoma (Bergmanm Y. and J. Haimovich. 1977. Characterization of acarcinogen-induced murine B lymphocyte cell line of C3H/eb origin. J.Immunol. 7:413-417) was obtained from R. Levy (Stanford, Calif.). The38C-13 tumor secretes and expresses IgM (κ) on the cell surface.Inoculation of as few as 10² 38C-13 tumor cells i.p. into normalsyngeneic mice results in progressive tumor growth and death of the hostwith a median survival time of only two weeks. Mice surviving past 60days from tumor challenge are long-term survivors. The BALB/c A20lymphoma (Kim, K. J., L. C. Kanellopoulos, R. M. Merwin, D. H. Sachs,and R. Asofsky. 1979. Establishment and characterization of BALB/clymphoma lines with B cell properties. J. Immunol. 122:(2)549-554) wasobtained from the American Type Culture Collection (Rockville, Md.) andexpresses IgGk. 38C-13 and A20 cells from a common frozen stock werepassaged in vitro 3 days before use in RPMI 1640 supplemented with 100U/ml of penicillin and streptomycin, 2×10⁻⁵ M 2-mercaptoethanol, andheat inactivated 10% fetal bovine serum (Gibco BRL, Gaithersburg, Md.).

In vivo immunizations and tumor protection experiment. Animal care wasprovided in accordance with the procedures outlined in a Guide for theCare and Use of Laboratory Animals (NIH Publication No. 86-23, 1985).Six- to nine-week old female C3H/HeNCrlBR or BALB/c mice (Charles RiverLaboratories, Frederick, Md.) were used. Syngeneic C3H/HeN or BalbC mice(10 per group) were immunized with Helios Gene Gun System (Bio-Rad,Hercules, Calif.) with plasmid DNA three times every two weeks. Theabdominal area of mice was shaved, and 1μ gold particles (Bio-Rad,Hercules, Calif.) carrying 1-3 μg DNA were injected at 400 psi. Twoweeks after the last immunization, mice were challenged i.p. with 200038C-13 lymphoma cells from a single preparation of tumor and followedfor survival. Differences in survival between groups were determined bynon-parametric logrank test (BMDP statistical software, Los Angeles).P-values refer to comparison with group immunized with DNA expressingthe same chemokine or defensin fused with an irrelevant sFv, or sFvfused with mutant chemokine, unless specified.

Therapy of established tumor with DNA vaccine. Six- to nine-week oldfemale BALB/c mice (ten per group) were challenged with 2.5×10⁵syngeneic A20 tumor cells. At day 1, 4, 8 and 18 these mice weregene-gun immunized with DNA plasmid (containing about 1-2 μg DNA perimmunization) and mice followed for tumor progression.

Results

Property of constructs: murine β-defensins and viral chemokines retaintheir functional integrity when produced as fusion proteins with sFv.First, a variety of chemokine and β3-defensin fusion proteins with sFv,a lymphoma Ig-derived non-immunogenic Fv, were cloned and purified(Table 3). The functional integrity of these proteins were tested by theability to induce chemotaxis of murine APC and THP-1 cells. As expected,chemokine fusion proteins induced dose-dependent chemotaxis. THP-1 cellswere chemo-attracted to vMIP1, human MCP-3, and SDF-1β fusion proteins,but not to the fusion with antagonist chemokine vMIP2, vMIP2sFv38.Control mutant fusion proteins generated for the each chemokine byreplacing the first Cys residue by Ser or by truncation of theamino-termini, as expected, did not induce chemotaxis of THP-1 cells ormurine DC. Furthermore, vMIP2 fusion proteins were tested for theirability to bind to their respective receptor (−s). vMIP2sFv38 coulddisplace labeled MIP1β and SDF1α in a dose dependent manner from CCR5and CXCR4 transfected cell lines, respectively. In contrast, nodisplacement was detected by vMIP2MsFv38 fusion protein, which containeda replacement Cys/Ser mutation in vMIP2 or by control sFv protein alone.

Since human β-defensin 2 was reported to act via CCR6, murine β-defensinfusion proteins were assayed for their ability to induce chemotaxis ofdifferent subsets of murine cells. β-defensin fusion proteins inducedchemotaxis of murine bone marrow derived iDC in a dose dependent mannerwith peak activity at 10 ng/ml and 100 ng/ml for Def2βsFv38 andDef3βsFv38, respectively. The immature phenotype of these DC (seeMethods) was also supported by their ability to migrate to human MIP3α,a chemo-attractant specific for CCR6⁺ immature DC (Dieu, M. C., B.Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Ait-Yahia, F.Briere, A. Zlotnik, S. Lebecque, and C. Caux. 1998. Selectiverecruitment of immature and mature dendritic cells by distinct

TABLE 3 Ligand-Antigen fusion constructs DNA vaccine Ligand: Defensin orname chemokine Antigen Protein Name Description Antigen alone psFv38none sFv38 sFv38 Single chain antibody fragment from 38C-13 lymphomapsFv20 none sFv20 sFv20 Single chain antibody fragment from A20 lymphomapgp120 none gp120 gp120 gp120 antigen (HIV-1, isolate 89.6) Defensinfusions: pmDF2βsFv38 murine β-defensin 2 sFv38 mDF2βsFv38 Murineβ-defensin 2 fusion with sFv38 pmDF2βgp120 murine β-defensin 2 gp120mDF2βgp120 Murine β-defensin 2 fusion with gp120 (HIV-1, 89.6)pmDF3βsFv38 murine β-defensin 3 sFv38 mDF3βsFv38 Murine β-defensin 3fusion with sFv38 pproDF2βsFv38 murine pro-β-defensin 2 sFv38mproDF2βsFv38 Murine pro-β-defensin 2 fusion with sFv38 Viral chemokineFusions: pvMIP2sFv38 viral MIP2 sFv38 vMIP2sFv38 Viral MIP2 fusion withsFv38 pvMIP1sFv38 viral MIP1 sFv38 vMIP1sFv38 Viral MIP1 fusion withsFv38 pMC148sFv38 MC148 sFv38 MC148sFv38 Viral MC148 fusion with sFv38Pro-inflammatory Chemokine fusions: phMCP3sFv38 Human MCP-3 sFv38hMCP3sFv38 Human MCP-3 fusion with sFv38 phMCP3sFv20 Human MCP-3 sFv20hMCP3sFv20 Human MCP-3 fusion with sFv20 phMCP3gp120 Human MCP-3 gp120hMCP3gp120 Human MCP-3 fusion with gp120 (HIV-1, isolate 89.6)phMDCsFv38 Human MDC sFv38 hMDCsFv38 Human MDC fusion with sFv38phMDCsFv20 Human MDC sFv20 hMDCsFv20 Human MDC fusion with sFv20phMDCgp120 Human MDC gp120 hMDCgp120 Human MDC fusion with gp120 (HIV-1,isolate 89.6) phSDF1βsFv38 Human SDF-1β sFv38 hSDF1βsFv38 Human SDF1-βfusion with sFv38 Mutant chemokine fusions: pvMIP2MsFv38 Mutant vMIP2sFv38 vMIP2MsFv38 Mutant vMIP2 fusion with sFv38 pvMIP1MsFv38 MutantvMIP1 sFv38 vMIP1MsFv38 Mutant vMIP1 fusion with sFv38 phMCP3MsFv38Mutant hMCP-3 sFv38 hMCP3MsFv38 Mutant human MCP-3 fusion with sFv38phMDCMsFv38 Mutant hMDC sFv38 hMDCMsFv38 Mutant human MDC fusion withsFv38 Control fusions: pmDF3βMuc1 β-defensin 3 Muc-1 mDF3βMuc1 Murineβ-defensin 3 fusion with 80 aa hMuc-1 peptide phMCP3-EGFP Human MCP-3EGFP hMCP3-EGFP Human MCP-3 fusion with EGFP protein phMDC-EGFP HumanMDC EGFP hMDC-EGFP Huma MDC fusion with EGFP protein Protein vaccine:Ig38-KLH None Ig38 Ig38-KLH 38C-13 lymphoma derived IgM proteincross-linked with KLH Ig20-KLH none Ig20 Ig20-KLH A20 lymphoma derivedIgG2a protein cross-linked with KLHchemokines expressed in different anatomic sites. J. Exp. Med.188:(2)373-386; Yang, D., O. M. Howard, Q. Chen, and J. J. Oppenheim.1999. Cutting edge: immature dendritic cells generated from monocytes inthe presence of TGF-beta 1 express functional C-C chemokine receptor 6.J. Immunol 163:(4)1737-1741), and their inability to react to humanMIP3β (Sallusto, F., B. Palermo, D. Lenig, M. Miettinen, S. Matikainen,I. Julkunen, R. Forster, R. Burgstahler, M. Lipp, and A. Lanzavecchia.1999. Distinct patterns and kinetics of chemokine production regulatedendritic cell function. Eur. J. Immunol 29:(5)1617-1625), achemo-attractant specific for CCR7+ mature DC. None of the defensinfusion proteins stimulated chemotaxis of mature DC, which migrated toMIP3β. Control fusion protein proDef2βsFv38 did not induce chemotaxis ofany DC. Therefore, murine β-defensin 2 and 3 fusion proteins canspecifically target immature DC. Furthermore, sFv fusion with β-defensinand xenogeneic human or viral chemokines does not disrupt theirfunctional chemokine properties.

Murine β-defensin, xenogeneic proinflammatory and viral chemokine fusionconstructs render non-immunogenic tumor antigen immunogenic. Next, thesefusion proteins were used to induce specific immunity againstnon-immunogenic sFv38 when administered as a DNA vaccine in mice. Tenmice per group were immunized by gene-gun with plasmids encoding fusionproteins with mature β-defensins, pDef2βsFv38 and pDef3βsFv38,respectively, or with human or viral chemokines, pSDF1βsFv38, pMDCsFv38,pvMIP1sFv38 or pvMIP2sFv38. Control mice were immunized with DNAconstructs encoding sFv fused with inactive pro-Defensin(pproDef2βsFv38), or mutated chemokines (phMCP3MsFv38, pMDCMsFv38,pMIP1MsFv38 and pvMIP2MsFv38). Mice immunized with plasmids encoding sFvfusion proteins with both murine β-defensins, murine MCP-3, human MDC orviral chemokines induced significant idiotype-specific antibodies whichwere comparable to the levels induced by vaccination with tumor-derivedintact Ig protein conjugated to KLH. In contrast, control mice immunizedwith an inactive pro-β-defensin (pproDef2βsFv38) or mutant chemokine sFvfusion constructs (hpMCP3MsFv38, pMDCMsFv38 and pvMIP2MsFv38), or sFv38alone did not produce any anti-Id antibody responses. Moreover, amixture of separate plasmids encoding β-defensin 3 (pmDF3βMuc1T) and sFvantigen (sFv38) failed to elicit a specific humoral response,demonstrating a requirement for sFv to be physically linked toβ-defensin or chemokine moiety. Therefore, non-immunogenic sFv wasrendered immunogenic by fusion with mature murine β-defensins, syngeneicmurine, xenogeneic human or viral chemokines. These results also suggestthat it was not sufficient to simply attract APC to the site ofproduction of sFv, but that direct APC targeting with self-antigen fusedto β-defensin or chemokine was required. Thus, induction of anti-Idantibodies by sFv fusion proteins appeared to involve receptor-mediatedbinding and delivery of antigen to APC. Vaccination elicited anti-Idantibodies of various isotypes, suggesting activation of differenteffector cells by different pro-inflammatory moieties. All carriersinduced specific IgG1 antibodies. However, MDC containing vaccines alsoelicited high titers of IgG2b and IgG3, while fusion constructs withβ-defensin-3 and vMIP2 elicited high titers of IgG2b but little IgG3,and little IgG2b or IgG3, respectively.

Two weeks after the last of three serial immunizations, mice werechallenged with a 20-fold lethal dose of syngeneic tumor. No survivalwas observed in control groups immunized with PBS or plasmids encodingsFv38 fused with inactive pro-β-defensin-2 (pproDef2βsFv38), MDC alone(pMDC-EGFP), or with mutant constructs pMDCMsFv38, pvMIP2MsFv38,pvMIP1MsFv38. Moreover, no protection was observed in mice immunizedwith DNA encoding a mixture of unlinked functionally active β-defensinor chemokine with sFv. In contrast, significant protective immunity waselicited in mice immunized with pDef2βsFv38 and pDef3βsFv38 (logrankP<0.001 as compared with pproDef2βsFv38 and pMIP2MsFv38, respectively).Similarly, pMDCsFv38, pvMIP2sFv38 and pvMIP1sFv38 immunized micedemonstrated statistically significantly prolonged survival (logrankP<0.001 as compared with PMDC-EGFP and mutant pvMIP2MsFv38 andpvMIP1MsFv38, respectively). Therefore, β-defensins and xenogeneic humanand viral chemokines can render a non-immunogenic self-tumor antigen(sFv) immunogenic and elicit specific antitumor immunity. These dataalso suggest that chemokine receptor engagement with chemokine- ordefensin-sFv fusion is useful for the induction of immunity.

Requirement for secretion but not for chemotaxis for induction of immuneresponses. Additional constructs were used for testing in vivo. Micewere immunized with MCP-3 fusion constructs with or without a secretoryleader sequence (pMCP3sFv38 and pMCP3sFv38-w/o-SL, respectively). Highlevels of Id-specific antibodies were detected in mice immunized withpMCP3sFv38, containing an intact secretory leader; however, noantibodies were elicited in mice immunized with pMCP3sFv38-w/o-SL.Furthermore, tumor protection was elicited only in those mice immunizedwith pMCP3sFv38, but not with pMCP3sFv38-w/o-SL (logrank P<0.001). Inaddition, no protection was detected in mice immunized with DNAexpressing a secretable but mutated MCP-3 fusion protein, which couldnot bind the respective receptor (pMCP3MsFv38). These data areconsistent with immunity being induced by APC which took up (viachemokine receptor) a functionally active chemokine fusion protein whichwas secreted from bystander cells, rather than by APC directly beingtransduced by gene gun immunization.

It was determined whether activation of receptor-mediated chemotaxis wasrequired for eliciting immune responses, or whether receptor bindingalone was sufficient. Immune responses of a selective pair of agonist(vMIP1) (Endres, M. J., C. G. Garlisi, H. Xiao, L. Shan, and J. A.Hedrick. 1999. The Kaposi's sarcoma-related herpesvirus (KSHV)-encodedchemokine vMIP-I is a specific agonist for the CC chemokine receptor(CCR)8. J. Exp. Med. 189:(12)1993-1998) and antagonist (MC148)(Luttichau, H. R., J. Stine, T. P. Boesen, A. H. Johnsen, D. Chantry, J.Gerstoft, and T. W. Schwartz. 2000. A highly selective CC chemokinereceptor (CCR)8 antagonist encoded by the poxvirus molluscumcontagiosum. J. Exp. Med. 191:(1)171-180) chemokines, which bind toCCR8, were compared to immune responses in mice immunized with plasmidsencoding pvMIP1and MC148 fusion proteins, respectively (pMIP1sFv38 andpMC148sFv38). Mice immunized with either of these chemokine fusionconstructs produced comparable levels of specific antibodies. Moreover,significant tumor protection was detected in both groups of micechallenged with a high dose of 38C-13 cells (logrank P<0.001 forpMC148sFv38 and P<0.01 for pvMIP1sFv38, respectively, compared withcontrol pvMIP1MsFv38). These data suggest that chemokine receptorbinding alone, in the absence of subsequent signaling for chemotaxis, issufficient to induce immunity.

Discussion

This example demonstrates that use of proinflammatory factors of innateand adaptive immunity such as β-defensins 2 and 3 and viral chemokines(Luster, A. D. 1998. Chemokines—chemotactic cytokines that mediateinflammation. N. Engl. J. Med. 338:(7)436-445) can help to elicit strongimmune responses both against a model non-immunogenic tumor antigen,lymphoma idiotype (Stevenson, F. K., D. Zhu, C. A. King, L. J. Ashworth,S. Kumar, and R. E. Hawkins. 1995. Idiotypic DNA vaccines against B-celllymphoma. Immunol. Rev. 145:211-228), and viral antigen, HIV gp120. Theappeal of this approach was based not only on the ability of thesemediators of innate and adaptive immunity to target surface receptors onAPC, particularly on iDC (Yang, D., O. Chertov, S. N. Bykovskaia, Q.Chen, M. J. Buffo, J. Shogan, M. Anderson, J. M. Schroder, J. M. Wang,O. M. Howard, and J. J. Oppenheim. 1999. Beta-defensins: linking innateand adaptive immunity through dendritic and T cell CCR6. Science286:(5439)525-528), presumably resulting in increased uptake of antigen,but possibly also to induce of expression of co-stimulatory moleculesand, in turn, production of other pro-inflammatory cytokines andfactors. This example demonstrates that both murine β-defensin 2 and 3efficiently induced chemotaxis of immature, but not mature, murine bonemarrow derived DC, suggesting that a β-defensin specific receptor(s) isexpressed on immature DC.

Example 6

As an example of how the vaccine of this invention can be administeredto a patient to treat cancer or to treat or prevent HIV infection (withthe additional administration of adjuvants, such as immunostimulatorycytokines, if desired), the following is a complete protocol for aclinical trial describing the administration of Id-KLH and GM-CSF topatients to treat follicular lymphoma. The same study design can beemployed for the administration of the defensin-tumor antigen fusionpolypeptide or the defensin-viral antigen fusion polypeptide of thepresent invention or nucleic acids encoding the fusion polypeptides ofthis invention, with appropriate modifications, as would be apparent toone of skill in the art. In particular, studies to test the efficacy ofHIV vaccines are well known in the art and the clinical protocoldescribed herein can be readily modified by one of skill in the art asappropriate to test the efficacy of the HIV fusion polypeptide or HIVfusion polypeptide-encoding nucleic acid of this invention according towell known protocols for testing HIV vaccines (126, 127).

Background and Rationale

Immunoglobulin (Ig) molecules are composed of heavy and light chains,which possess highly specific variable regions at their amino termini.The variable regions of heavy and light chains combine to form theunique antigen recognition site of the Ig protein. These variableregions contain determinants that can themselves be recognized asantigens, or idiotopes. B-cell malignancies are composed of clonalproliferations of cells synthesizing a single antibody molecule withunique variable regions in the heavy and light chains. B-cell lymphomasare neoplasms of mature resting and reactive lymphocytes which generallyexpress synthesized Ig at the cell surface. The idiotypic determinantsof the surface Ig of a B-cell lymphoma can thus serve as atumor-specific marker for the malignant clone.

Studies in experimental animals, as well as in man, have demonstratedthe utility of the Ig idiotype as a tumor-specific antigen for the studyof the biology of B-cell lymphoma in vitro and as a target for passiveimmunotherapy in vivo (1, 2, 3). Furthermore, active immunizationagainst idiotypic determinants on malignant B cells has beendemonstrated to produce resistance to tumor growth in a number ofsyngeneic experimental tumor models, as well as specific anti-tumortherapy against established tumors (4-13). These results, takentogether, provided the rationale for testing autologous tumor-derivedidiotypic surface Ig (Id) as a therapeutic “vaccine” against humanB-cell lymphoma. Furthermore, preclinical studies in subhuman primatesdemonstrated that optimal immunization with human lymphoma-derived Idrequired conjugation of the protein to an immunogenic protein carrier(keyhole limpet hemocyanin; KLH) and emulsification in an adjuvant (14).

Guided by these observations, nine patients with B-cell lymphoma wereimmunized with autologous Id protein (15). These patients received noanti-tumor therapy during the time of the study. They were either incomplete remission or in a state of minimal residual disease followingconventional chemotherapy. In addition, three patients with rapidlyprogressive recurrent lymphoma were enrolled in a separate safety study;all three required reinstitution of chemotherapy shortly afterenrollment, did not complete the immunization series, and were notstudied further. They received intramuscular injections of 0.5 mg of Idconjugated to KLH at 0, 2, 6, 10 and 14 weeks, followed by two boosterinjections at 24 and 28 weeks. Patients in the first trial (fivepatients) received Id-KLH alone for the first three immunizations, thenId-KLH emulsified in a Pluronic polymer-based adjuvant vehicleformulation for all subsequent immunizations. Because noidiotype-specific immune responses were observed prior to the additionof the adjuvant to the program in this first group of patients, patientsin the second trial (four patients) received the entire series ofimmunizations with this adjuvant. All patients were analyzed foridiotype-specific antibody production and peripheral blood mononuclearcell (PBMC) proliferative responses in vitro immediately before eachimmunization and at one to two month intervals following the lastimmunization. The KLH carrier provided a convenient internal control forimmunocompetence of the patients and all patients demonstrated bothhumoral and PBMC proliferative responses to the KLH protein, with theexception of one patient, who demonstrated only the latter. Seven of thenine patients demonstrated either a humoral (n=2) or a cell-mediated(n=4) anti-idiotypic immunological response, or both (n=1).

Anti-idiotypic antibody responses were detected by analysis of pro- andhyper-immune sera in either direct, or competition, ELISA. Theimmunization with autologous Id protein induced significant titers ofanti-idiotypic antibody that either directly bound or inhibited thebinding of a murine anti-idiotype monoclonal antibody (anti Id mAb) toId on the plate. The specificity of the humoral response for the Igidiotype was demonstrated by the lack of significant binding ofhyperimmune serum to a panel of isotype-matched human Igs of unrelatedidiotype, or by the lack of significant inhibition of a panel ofheterologous Id-anti-Id systems, respectively. Peak humoral responseswere obtained after the fifth immunization and persisted for at leastnine months. The anti-idiotypic antibody produced by patient 1 wasaffinity-purified and shown to contain heterogeneous light chains aswell as immunoglobulin G heavy chains. This patient's antibody titer wassuccessfully boosted with a single administration of Id-KLH in adjuvantafter a decline of the humoral response after 15 months.

Cellular immune responses were measured by the proliferation of PBMC toKLH and to autologous Id separately at concentrations ranging from 1-100μg per milliliter of soluble protein in five day in vitro cultures. Noneof the pre-immune PBMC demonstrated any preexisting proliferation toautologous Id above that to culture medium alone. Hyperimmune PBMC fromall patients demonstrated strong proliferative responses to the KLHcarrier. Of primary interest, significant hyperimmune proliferativeresponses to Id were detected in five patients. Although their responseswere of lower magnitude than parallel responses to KLH, patients 3, 4,6, 8 and 9 were classified as responders on the basis of reproducibleincreases in counts-per minute (cpm) ³H-thymidine incorporation in wellscontaining Id, compared with medium alone, that were sustained overmultiple time points. Patients demonstrating occasional increases in cpmin wells containing Id compared with medium alone were classified asnon-responders (patients 1 and 5).

Flow cytometry analysis of cultures demonstrating proliferation to Idrevealed a predominance of cells staining positively for CD4 (>95%),suggesting the phenotype of the responding cell subpopulation. Thesecultures could be successfully expanded for approximately four weeks bystimulation alternatively with interleukin-2 (IL-2) and Id-pulsedautologous irradiated PBMC as antigen-presenting cells. Specificity ofthe responses for Ig idiotype was confirmed by the lack of significantproliferation to an isotype-matched human Ig of unrelated idiotypecompared with medium alone. Such idiotype-specific PBMC proliferativeresponses were observed only after the addition of the adjuvant to theprogram and also persisted for at least 9-14 months.

The ability of the idiotype-specific humoral response to bind autologoustumor cells was also tested. This was shown by the inhibition of bindingof a labeled murine anti-idiotype mAb to tumor cells from apre-treatment lymph-node specimen from patient 8 by hyperimmune, but notby pre-immune, serum from this patient. In addition, affinity purifiedanti-idiotypic antibodies from the hyperimmune sera of the two otherpatients who demonstrated idiotype specific humoral responses weredemonstrated by flow cytometry to bind autologous tumor.

All patients were also closely monitored for disease activity withphysical examinations and routine laboratory and radiographic studies.Of the two patients with measurable tumor at the initiation of Idimmunization, one (patient 1) experienced complete regression of asingle 2.5 cm left submandibular lymph node, and the other (patient 4)experienced complete regression of a 4.5 cm cutaneous lymphomatous masson the right arm. This clinical response in patient 4 correlated with anId-specific, PBMC proliferative response in vivo. Correlating with theduration of their immunological responses, the clinical responses inboth patients have continued at 24 and 10 months, respectively, aftercompletion of the immunization series. Moreover, with a median follow uptime of 10 months, the only case of tumor recurrence among thosepatients who were in remission and completed the immunization seriesoccurred in patient 5, who was one of the two patients who failed todemonstrate an idiotype-specific immunological response.

Toxicity was minimal in all twelve patients. All patients experiencedtransient local reactions characterized by mild erythema, induration,and discomfort, without skin breakdown, at the injection sites.Splitting the components of the vaccine (Id-KLH and adjuvant) in onepatient who had experienced a moderate local reaction and in anotherpatient who had experienced a moderate systemic reaction, characterizedby fever, rigors and diffuse arthralgias, established the adjuvant asthe component associated with these reactions. Both of these moderatereactions resolved completely after 24-48 hours. The only laboratoryabnormality associated with Id immunization was a mild elevation (lessthan twice the normal value) of serum creatine phosphokinase 24 hoursafter immunization in an occasional case.

These results demonstrate that patients with B-cell lymphoma can beinduced to make sustained idiotype-specific immune responses by activeimmunization with purified autologous tumor-derived surface Ig. Theyshow that autologous Id, made immunogenic by conjugation to KLH, canserve as an immunogen (antigen) to elicit host immunological responses.The induction of low levels of idiotype-specific immunity wasdemonstrated in the setting of minimal tumor burden followingconventional chemotherapy. These results, taken together with theinduction of relatively stronger immune responses to the KLH carrier,and exogenous antigen, suggest that chemotherapy-inducedimmunosuppression is not an obstacle to active immunotherapyadministered adjunctively to cytoreductive drug therapy in this manner.

This initial study also established the requirement for an immunologicaladjuvant, as no Id-specific responses were observed prior to theaddition of an adjuvant to the program. The objective of furtherclinical trials using tumor derived Id as a therapeutic vaccine is tofurther optimize the immunogenicity of this vaccine. To this end, thisstudy will focus on the use of novel immunological adjuvants whichare 1) more potent and 2) more effective in the induction ofcell-mediated immune responses, compared with the pluronic polymer-basedadjuvant used in the study.

The 38C13 B cell tumor is used as a model system to screen promisingimmunological adjuvants. A number of these have included cytokines andamong these, GM-CSF has emerged as a promising adjuvant for idiotypic Igantigen. In these experiments (10 mice per group), syngeneic mice wereimmunized with 50 μg Id-KLH derived from the tumor, either alone or incombination with GM-CSF mixed together with the antigen and administeredsubcutaneously. Three additional daily doses of GM-CSF were administereds.c. as close to the original site of immunization as possible. Miceimmunized with an irrelevant Id-KLH (4C5 IgM) served as negativecontrols for the vaccine. Two weeks after this single immunization, allmice were challenged with a single preparation of 38C13 tumor cells(5×10³ cells i.p.) and followed for survival. The results demonstratedthat the augmented survival benefit afforded by immunization withrelevant Id-KLH alone can be significantly enhanced by the addition ofGM-CSF at either the 100 or 10,000 unit dose. The loss of thisprotective effect at a higher dose of GM-CSF of 50,000 units was alsoobserved. These data suggest that GM-CSF may have a potent adjuvanteffect in vivo for Id-KLH antigen, especially at relatively low doses.

The follicular lymphomas are follicular small cleaved cell (FSC) andfollicular mixed lymphoma (FM). Stage I and II patients comprise only10% to 15% of all cases of follicular lymphomas and are best managedwith radiation therapy. Eight-five percent of patients with follicularlymphomas present with stage III or IV disease. The optimal managementof these patients remains controversial and has generally followed twodivergent approaches (16, 17). One is an aggressive approach, which hasincluded radiation therapy, combination chemotherapy, or combinedmodality therapy and the other is a conservative approach that involvesno initial treatment followed by a single-agent chemotherapy orinvolved-field radiotherapy when required (18; 19). Most forms ofsystemic therapy have the capacity to produce high complete responserates. However, they have failed to produce long-term disease-freesurvival or to prolong overall survival; thus, it has become clear thatthe vast majority of patients with this disease will relapse and die oftheir lymphoma, despite its usually indolent course.

The NCI study (MB-110, BRMP 8903) begun in 1978, is a prospectiverandomized study comparing these two distinct approaches to themanagement of stage ImI or IV indolent histology lymphoma. Most patientswere randomized between no initial therapy or aggressive combinedmodality therapy with ProMACE/MOPP flexitherapy followed by low dose(2400 cGy) total nodal irradiation. Among the 149 patients treated thusfar, 125 (84%) were randomized; 62 to watch and wait (W & W) and 63 toaggressive treatment. Among the 62 patients on the watch and wait arm,29 continue to be observed for periods up to 10+ years. The median timeto cross over to aggressive therapy is 23 months.

It is apparent that patients in whom therapy is initiated after thedevelopment of symptoms have a significantly lower complete responserate to therapy than patients randomized to receive the same therapy atdiagnosis (74% vs 40%, P₂=0.0039). The complete responder (CR) rate ofpatients randomized to initial aggressive treatment is comparable tothose obtained in patients with advanced-stage intermediate gradelymphoma receiving the same treatment. The CR rate in indolent lymphomadoes not appear to be significantly higher than what can be achievedwith other combination regimens. For patients randomized to watch andwait, median follow-up of CRs is shorter because of the delay ininitiating treatment. However, the median duration of remission has notbeen reached at five years and 57% of patients are projected to bedisease-free>8 years and 44% are projected to be in a CR at 12 years.The disease-free survival curves are not significantly different betweenthe two arms. Thus, allowing the patient to reach a greater tumor burdenbefore instituting systemic therapy reduces the likelihood of obtaininga CR, but once achieved, CRs are comparably durable to those obtainedfrom primary aggressive therapy. The lengthening of the remissionduration, however, has not resulted in a survival advantage for patientsrandomized to receive primary aggressive chemotherapy. Furthermore, eventhough a minority of complete responders have relapsed, the probabilityof relapse appears to be continuous over time, and the vast majority ofpatients are expected to eventually succumb to their disease.

Thus, even immediate aggressive therapy has not resulted in improvedsurvival. Therefore, although patients diagnosed with follicularlymphoma enjoy relatively longer survival times compared with patientswith solid tumors, follicular lymphoma remains an incurable disease.Novel experimental therapies designed to improve the durability of theremissions already effectively induced by chemotherapy are justified.

Summary of Treatment Plan

The goal is to treat patients with follicular lymphomas to completeremission or maximal response with ProMACE chemotherapy. After thecompletion of chemotherapy, in an effort to reduce the relapse rate (byeradicating microscopic disease resistant to chemotherapy), patientswill receive an autologous Id vaccine administered in combination withGM-CSF.

The goal of this study is to evaluate the ability of the Id vaccine toclear the bone marrow of malignant cells detectable by pathologic(morphologic) examination or molecular examination (polymerase chainreaction, PCR) in patients with PCR amplifiable translocations. Allpatients have serial bone marrow and peripheral blood samples collectedto search for clonal abnormalities by PCR. Patients are followed aftervaccine therapy and their remission status correlated with clinical vs.molecular determinations of response. There should be three categoriesof complete responders: those who had a clinical complete responsebefore the vaccine but had an abnormal clone by PCR that cleared afterthe vaccine; those with a clinical CR before the vaccine who were alsoPCR negative before the vaccine; and those who achieved a clinicalcomplete response but had PCR positive marrows before and after thevaccine. It is a goal of this study to assess whether “molecularcomplete responses” can be achieved using the vaccine in patientsfollowing chemotherapy.

Objectives

The objectives of this trial are to:

To induce cellular and humoral immunity against the unique idiotypeexpressed on the surface of patients' B-cell lymphomas.

To determine the ability of Id immunization to eradicate bcl-2 positivetumor cells from the bone marrow as detected by PCR.

As a secondary objective, to determine the more biologically active ofthe two GM-CSF doses as an adjuvant, as measured by the endpoints in theabove objectives.

To determine the impact of Id immunization on disease free survival ofpatients achieving a CR with chemotherapy.

Patient Selection

Patient Sample

A. Sample size, approximately 42 patients

B. Sex distribution: male and female

C. Age: patients must be ≧18 years old

Eligibility Criteria

-   -   Patient must meet all of the following eligibility criteria:    -   A. Tissue diagnosis of: follicular small cleaved cell, or        follicular mixed lymphoma with surface IgM, IgG or IgA phenotype        with a monoclonal heavy and light chain. Pathology slides must        be submitted to the NIH Pathology Department for review.    -   B. Stage III or IV lymphoma.    -   C. Only previously untreated patients are eligible.    -   D. Previous treatment with radiation alone (less than TBI) is        permissible.    -   E. A single peripheral lymph node of at least 2 cm size        accessible for biopsy/harvest.    -   F. Karnfsky status≧70%.    -   G. Life expectancy of >one year.    -   H. Serum creatinine≦1.5 mg/dl unless felt to be secondary to        lymphoma.    -   I. Bilirubin≦1.5 mg/dl unless felt to be secondary to lymphoma        or Gilbert's disease. SGOT/SGPT<3.5× upper limit of normal.    -   J. Ability to give informed consent. Ability to return to clinic        for adequate follow-up for the period that the protocol        requires.

Patient Exclusion Criteria

-   -   The presence of any exclusion criteria (listed below) will        prohibit entry into study:    -   A. Prior total body irradiation.    -   B. Presence of antibodies to HIV, hepatitis B surface antigen or        other active infectious process.    -   C. Pregnancy or lactation. Fertile men and women must plan to        use effective contraception. A beta-HCG level will be obtained        in women of childbearing potential.    -   D. Patients with previous or concomitant malignancy, regardless        of site, except curatively treated squamous or basal cell        carcinoma of the skin, or effectively treated carcinoma in situ        of the cervix.    -   E. Patient unwilling to give informed consent.    -   F. Failure to meet any of the eligibility criteria described        above.    -   G. Any medical or psychiatric condition that in the opinion of        the protocol chairman would compromise the patient's ability to        tolerate this treatment.    -   H. Patient with CNS lymphoma (current or previously treated)        will not be eligible.        Clinical Evaluation

Complete history and physical examination.

CBC, diff., platelet count.

Serum chemistry, β₂-microglobulin.

PT/PTT

Quantitative immunoglobulins, serum protein electrophoresis,immunoelectrophoresis.

HIV antibody, HBsAg.

Urinalysis.

Serum β-HCG in women of child-bearing potential.

EKG and MUGA.

5 TT for serum storage.

Leukapheresis to obtain 3×10⁹ lymphocytes. These samples will be usedfor baseline studies of T-call activation and response to Id.

Tumor Biopsy—prior to therapy, all patients must undergo biopsy/harvestof a clinically involved peripheral lymph node to obtain tissue formorphological classification, immunophenotypic characterization,determination of immunoglobulin gene rearrangements, bcl-2translocation, cytogenetics, and to provide starting material for an Idvaccine. The sample should be at least 2 cm in size. Only patients withtumors that are surface immunoglobulin positive with a monoclonal heavyand light chain will be accepted as study candidates. Use standardlymphoma vaccine biopsy orders (see protocol below). Leftover tumorbiopsy samples may be used for basic studies of lymphoma biology invitro. Such future studies may be done without re-consenting thesubjects only if the studies involve risks already outlined in theoriginal consent form.

CXR-PA and LAT.

CT scan of abdomen and pelvis.

Lymphangiogram, unless contraindicated by massive pedal edema, severechronic lung disease, ethiodal sensitivity (Note: sensitivity to otheriodine compounds, e.g., renograffin, are relative, but not absolutecontraindications).

Other tests (CT chest, ultrasound, liver scan, bone scan, upper andlower GI series, IVP, MRI) should be performed as needed to evaluate alldisease sites adequately.

Examination of pleural fluid or ascites when present.

Bilateral bone marrow aspirates and biopsies—In addition to the normalaspirate and biopsy, 5 cc of marrow will be aspirated from each sideinto 0.5 ml of PFH for PCR analysis. The procedure should be performedin the usual manner with a biopsy performed first. Then a small volume(0.5-1 cc) can be aspirated for the smear and clot tube. A separateRosenthal needle with bevel should be used for the aspirate. The 5 ccsample for PCR can be obtained from the same site as the initialaspirate.

CT scan of the head and lumbar puncture with CSF analysis if clinicallyindicated.

Patient Registration

Patients will be registered prior to the initiation of therapy at whichtime eligibility criteria will be reviewed. Stratification andrandomization are described in detail below (see Statisticalconsiderations).

Study Design

ProMACE

Day 0 Day 7 Day 28 Cyclophosphamide Cyclophosphamide Next cycle begins650 mg/m² IV 650 mg/m² IV Doxorubicin Doxorubicin  25 mg/m² IV  25 mg/m²IV Etoposide VP-1 6 Etoposide BP-1 6 120 mg/m² IV 120 mg/m² IV

-   -   Prednisone 60 mg/m² po qd×14 (days 0 to 13)    -   Bactrim one double strength tablet po BID throughout therapy

All patients will be treated until a complete remission is obtained andtwo additional cycles of chemotherapy have been given, or until diseasehas been stable for two cycles of chemotherapy, or progressive diseasedevelops. A minimum of six cycles will be given to each completeresponder before therapy is discontinued. Patients with more than 90% PRor a full CR will be continued on the vaccination part of the protocol.Patients with less than 90% PR or progressive disease will be taken offof the study.

Postinduction Therapy—Three to six months (or whenever a customized GMPvaccine is available, up to a maximum period of 12 months) after thecompletion of chemotherapy, all patients in whom either a completeclinical remission or minimal disease status (≧90% partial response) hasbeen achieved will receive a series of five injections of a vaccineconsisting of 0.5 mg autologous tumor derived immunoglobulin (Id)conjugated to KLH. The vaccine will be administered together with GM-CSFas an immunological adjuvant. Both the vaccine and GM-CSF will beadministered subcutaneously according to the following schedule:

Schedule: At 0, 1, 2, 3 and 5 months

-   -   Id-KLH (0.5 mg s.c.) day 0    -   adjuvant (s.c.) days 0-3    -   Cohort 1: GM-CSF 500 mcg/m²/d s.c. for 4 days    -   Cohort 2: GM-CSF 100 mcg/m²/d s.c. for 4 days

The sites of injection will be rotated between the upper and lowerextremities. Each dose of vaccine or GM-CSF will be split equallybetween the two upper or lower extremities. All GM-CSF injections willbe given in close proximity to the vaccination site, as close to theexact site of injection as possible. If local reactions to GM-CSF aresevere, GM-CSF injections may be given elsewhere. Patients will beobserved in the clinic for two hours following Id-KLH and/or GM-CSFadministration. During the observation period, vital signs will be takenevery 15 minutes during the first hour and every 30 minutes during thesecond hour.

Supportive Care

G-CSF 5 mcg/kg/d SC may be used in all patients who are hospitalized forthe treatment of febrile neutropenia, regardless of how long theneutropenia persists.

Grading and Management of Toxicity

Chemotherapy: Dose modification of chemotherapy will be based on thegranulocyte count done at the time of drug administration (day 0 or 7 ofeach cycle). The percentage of drugs administered may be furthermodified based on toxicity in prior cycles (see below). If thegranulocyte count is <1200, and the patient is due for day 0 drugs,delay day 0 for one week until appropriate parameters are met. Ingeneral, delays of up to one week are preferable to starting G-CSF. Ifafter a one week delay, appropriate parameters are still not met, thenG-CSF may be started as above. Also, in general, delays of up to oneweek are preferable to dose reductions. Full doses of all drugs shouldbe given on time if blood count suppression is due to bone marrowinvolvement with disease.

Dose Modification for Hematologic Toxicity

IF GRANULOCYTE COUNT IS: THEN DOSE On Day 0 AS FOLLOWS: ≧1200 100% alldrugs ≦1200 Day 0 Delay

For neutrophil nadir<500 or platelet count<25,000 on previous cycle, 75%of cyclophosphamide, doxorubicin, and etoposide should be considered.For neutrophil nadir (day 21 counts)>750 on a previous cycle, doseescalation of cyclophosphamide, doxorubicin, and etoposide by 10-20%should be prescribed.

IF PLATELET COUNT IS: THEN DOSE AS FOLLOWS: >100,000 100% of all drugs50-99,999 100% Prednisone  75% Etoposide  50% Cyclophosphamide,Doxorubicin  <50,000 Delay

Dose Modification for Non-hematologic Toxicity

Assessment of non-hematologic toxicity will be graded according to theCRB/DCS/NCI Common Toxicity Criteria. Chemotherapy will be withheld inpatients experiencing grade 2 or greater non-hematologic toxicity untilthe patient has completely recovered from the toxicity. Fornausea/vomiting 2: grade 2, drug therapy should be continued withnon-steroid antiemetics.

Doxorubicin dosage should be adjusted as follows in the presence of thefollowing LFT abnormalities:

% Dose Bilirubin SGOT 100 <1.5 mg/dl <75 U 50 1.5-2.9 mg/dl 75-150 U 253.0-5.9 mg/dl 151-300 U 0 ≧6.0 mg/dl >300 U

Immunotherapy

-   -   Id-KLH Vaccine

Based on previous experience with autologous Id-KLH vaccines, little orno toxicity is expected from the Id-KLH component of the vaccine (15).Nevertheless, any local skin reactions will be carefully noted andscored for erythema, induration, pain and disruption of the barriersurface. If any patient has a reaction suggestive of sensitization, thevaccine may be split into its component parts; specifically, the patientwill be tested with Id-KLH alone and then GM-CSF alone. Toxicities willbe graded according to the CRBINCI/DCS common toxicity criteria.

-   -   GM-CSF

Anticipated toxicities from GM-CSF administration in this dose range areexpected to be mild based on previous experience. Potential toxicitiesinclude fever, chills, myalgias, arthralgias, nausea, vomiting,diarrhea, dyspnea, tachycardia, arrhythmias, elevation of liver functiontests, elevation of BUN and creatinine. However, local skin reactions,such as erythema and induration, may be observed and will be carefullynoted. Attempts will be made to maintain these patients as outpatients.For grade IV fever (not responsive to Indocin or Tylenol), or grade IIIvomiting (unresponsive to therapy), GM-CSF will be held until toxicityis less than grade II and will be restarted at 50% of the original doselevel for the rest of that weekly injection cycle and for subsequentcycles. For neurologic toxicity that affects daily function (unable tocarry on simple routine duties, or grade II in the toxicity gradingscale), hold treatment until symptoms resolve, then reduce GM-CSF by50%. If symptoms persist, the adjuvant should be removed for subsequentimmunizations. Patients with grade III neurotoxicity will be removedfrom the study.

For well-documented evidence of cardiac toxicity (i.e., grade III,including evidence of ischemia or ventricular arrhythmia, but notsupraventricular tachycardia or atrial fibrillation controlled bydigoxin or calcium channel blocking agents), the adjuvant will beremoved for subsequent immunizations.

Asymptomatic elevations in serum bilirubin and creatinine (not resultingin hyperkalemia) will be tolerated. For SGOT or SGPT>10× normal, GM-CSFwill be held until values return to <5× normal, then resumed at 50% ofthe GM-CSF dose for all remaining doses.

Fever and chills associated with vaccine administration and/or GM-CSFwill be treated with TYLENOL and/or DEMEROL. The use of non-steroidalantiinflammatory drugs and/or steroids should be avoided. Shouldnon-steroidals or steroids be required for unrelated medical conditionsfor a course exceeding 2 weeks, the patient will be taken off of thestudy.

Adverse Drug Reactions

All toxicities and adverse events will be recorded on the study flowsheet and appropriately graded as to severity and cause. Toxicities thatare related to the underlying disease should be clearly differentiatedfrom drug toxicities.

Adverse drug reactions related to chemotherapy will be submitted basedon guidelines for commercial drugs.

Reports of adverse reactions to Id-KLH and GM-CSF will be made using theDivision of Cancer Treatment Common Toxicity Criteria for referenceaccording to the guidelines published by the DCT, NCI. These guidelinescan be summarized as follows:

-   -   A. Report by telephone to IDB within 24 hours (301) 230-2330        -   1. All life-threatening events (grade 4, except for grade 4            myelosuppression) which may be due to administration of the            investigational drug(s),        -   2. All fatal events (grade 5),        -   3. All first occurrences of any previously unknown toxicity            (regardless of grade).    -   B. A written report should follow within 10 working days.    -   C. All adverse drug reactions will also be reported in writing        to the NCI Institutional Review Board within 10 working days.    -   D. All adverse drug reactions will also be reported to the FDA        in accordance with Federal regulations.    -   E. Data will be submitted at least every two weeks.        Study Parameters

During Chemotherapy

-   -   Weekly: CBC, diff. platelets; except day 14, i.e. CBC on day 0,        7, 21, and 28.    -   Beginning of each cycle: Chem 20, CXR, LAG follow-up (KUB), CT        scans (only after 4 cycles, then every 2 cycles).    -   Bilateral bone marrow aspirate and biopsy after four cycles and        every additional two cycles thereafter. Include 5 cc of aspirate        in PFH from each side for PCR analysis.

At Maximal Response to Chemotherapy

-   -   If residual disease is obvious, record measurements and perform        bone marrows as above.    -   For complete responders, complete restaging should be performed.        This should include all studies that were positive at initial        staging evaluation with the exception of repeat thoracotomy or        laparotomy. Bilateral bone marrows should be performed as above.

During Vaccine Therapy

-   -   If residual disease is obvious, record measurements and perform        bone marrows as above.    -   PT-PIT day 0    -   UA, β₂ microglobulin day 0 of each immunization.    -   Leukapheresis is performed on the day of initiation of vaccine        therapy (prior to the first cycle only) to obtain pre-vaccine        lymphocytes for storage. Five tiger top tubes are drawn at this        time to obtain serum for storage.    -   Two tiger top tubes and peripheral blood (60 cc in PFH) are        collected on day 0 of each monthly cycle, for preparation of        serum and lymphocytes, respectively.    -   Skin Biopsy is obtained near a planned immunization site on day        0 prior to the first cycle (baseline sample) and again on day 1,        2, or 3 of cycle 3 at an active site of erythema and/or        induration as close to the original biopsy site as possible.    -   DTH—Delayed type hypersensitivity test (DTH) to autologous        idiotype protein is performed during cycle 4 and again following        completion of the immunization regimen, i.e., during or after        cycle 5. The DTH-test is performed by intradermal injection of        0.5 mg of idiotype protein in 0.1-0.2 ml of NS. To ascertain the        specificity of a positive reaction, 0.5 mg of a heterologous        isotype matched Id-protein (from another patient on the same        study) in the same volume will be used as a negative control.        The control idiotypes used on these two occasions will be from        two different patients, also in the study, in order to minimize        the possibility of eliciting an immunologic response against a        particular irrelevant idiotype. A skin biopsy will also be        obtained at the site of the intradermal injection of idiotype        protein and at the control site, one to three days, after the        intradermal injections.    -   Fine needle aspiration or core biopsy (with or without CT        guidance) of any enlarged lymph node draining the vaccination        sites is performed to obtain lymphocytes for in vitro assays.

At Discontinuation of Vaccine

-   -   Restaging as described for Chemotherapy (see “At Maximal        Response to Chemotherapy,” above).    -   Bilateral bone marrow aspirates and biopsies at completion of        therapy and every six months for two years after completing        therapy and yearly thereafter.    -   10 cc of serum for storage and 60 cc of peripheral blood in PFH        is collected at completion of therapy and every three months for        a year.        Specimen Processing and Immunological Assays

Lymph Node Harvest/Biopsy

Each lymph node biopsy will be divided as follows: (a) one-third of thespecimen will be sent in saline to the Hematopathology Section,Laboratory of Pathology, NIH. Biopsies are processed for routinehistopathy and for immunophenotypic characterization, particularly withrespect to monotypic heavy and light chain expression; and (b)two-thirds of the specimen is sent in sterile saline in a sterilecontainer to Clinical Immunology Services, NCIFCRDC, where it isprocessed into a single-cell suspension and cryopreserved.

Blood and Bone Marrow Samples

All peripheral blood and bone marrow aspirate samples are sent in anexpedited manner to Clinical Immunology Services, NCI-FCRDC. Tiger toptubes are spun down and serum divided into 1 ml aliquots for frozenstorage. Peripheral blood mononuclear cells (PBMC) are isolated prior tofreezing by Ficoll-hypaque centrifugation using standard protocols.

Assay for Serum Antibody

In a direct enzyme-linked immunosorbent assay (ELISA), preimmune andhyperimmune serum samples from each patient are diluted over wells of amicrotiter plate that are coated with either autologous immunoglobulinidiotype or a panel of isotype-matched human tumor immunoglobulins ofunrelated idiotype. Bound antibody is detected with horseradishperoxidase-goat antihuman light-chain antibodies directed against thelight chain not present in the immunoglobulin idiotype (CaltagLaboratories, South San Francisco).

Assay for Idiotype-Specific Proliferative Response

Whenever feasible, fresh PBMC, isolated above, are used on the same daythey are obtained. Stored frozen PBMC are available as a back-up. PBMCare washed and plated at a concentration of 4×10⁵ cells per well inIscove's modified Dulbecco's medium (IMDM) with 1 percent human AB7serum (IMDM-1 percent AB). KLH, autologous immunoglobulin idiotype, or apanel of isotype matched immunoglobulins of irrelevant idiotypes atconcentrations of 0 to 100 μg per milliliter in IMDM-1 percent ABpreparation are added in triplicate. After the cells are incubated forthree days at 37° C. in an atmosphere containing 5 percent carbondioxide, they are transferred to a preparation of IMDM and 5 percentfetal-.calf serum containing recombinant interleukin-2 (30 Uppermilliliter). The plates are incubated for two days and pulsed for 16 to20 hours with 3H-labeled thymidine (1 μCi per well). Data are expressedas mean (±SEM) counts per minute of [³H]thymidine incorporation.

Initial five-day cultures of PBMCs established as described above areexpanded in IMDM-5 percent fetal-calf serum containing interleukin-2 (30Upper milliliter). Harvested cells are replaced in IMDM-1 percent ABcontaining autologous immunoglobulin idiotype and fresh irradiated (5000R) autologous PBMCs (4×10⁵ cells per well) as antigen-presenting cellsfor five days, before pulsing with ³[H]thymidine.

Cytotoxicity Assays

The potential cytotoxicity of PBMC cultured with Id as above, or withirradiated fresh cryopreserved tumor cells, is assayed against eitherautologous lymphoblastoid cell lines (LBL) pulsed with Id or freshcryopreserved tumor targets. Autologous LBL pulsed with soluble antigenhave been used successfully as targets to detect gp 160-specificcytotoxic T-lymphocytes (20). Historically, the inability to establishlong-term cultures of follicular lymphoma has hindered theiravailability as targets. However, two recent reports have described theuse of fresh cryopreserved lymphoma cell targets, with levels ofspontaneous incorporated radioisotope release in the acceptable range of<35% (21-22). Standard four hour ⁵¹Cr release, as well as 18-24 hour¹¹¹In release assays are used.

Autologous LBL are prepared from pre-immune PBMC by the AIDS MonitoringLaboratory, NCI-FDRDC, using published methods.

Monitoring of T-cell Receptor (TCR) Status

Pre-chemotherapy and pre- and postimmunization serum samples are assayedfor TCR status by Western blot assay. Approximately 7×10⁶ purifiedT-cells from PBMC are lysed for 5 minutes at 4° C. in lysis buffer (25mM Tris, pH 7.4 [Sigma Chemical Co., St Louis, Mo.], 300 mM NaCl, 0.05%Triton X-1 00, 1 mM Na orthovanadate, 10 μg/ml aprotinin, 10 μg/mlleupeptin, 10 mM nitrophenol-guanidine benzoate [NPGB] and 5 mM EDTA).The lysates are centrifuged at 12,000 rpm at 4° C. for 5 minutes andsupernatant is removed with a micropipettor, making sure the nuclearpellet is not disturbed. A sample of the supernatant is then used toquantitate protein using the BCA protein assay (Pierce, Rockford, Ill.).The rest of the lysate is boiled with 3× reducing sample buffer for 5minutes and placed on ice before its use in Western blot.

Varying concentrations of cellular lysate ranging between 1 and 30 μgare electrophoresed in 14% Tris-glycine gels (Novex ExperimentalTechnology, CA) under reducing conditions and then transferred toImobilon-p PVDF transfer membranes (Millipore Co., Bedford, Mass.). Themembranes are incubated with a 5% solution of non-fat dried milk for onehour and then blotted for one hour at room temperature with anti-TCRζanti-serum (Onco-Zeta 1, OncoTherapeutics, Cranbury, N.J.) at a 1:2000dilution. The membranes are washed with TBS-T buffer [1M Tris base, 5MNaCl, 0.1% Tween 20 (pH 7.5)] and incubated with anti-rabbit oranti-mouse Ig horseradish peroxidase (Amersham, Buckinghamshire, UK).After washing with TBS-T, the membranes are developed with thechemiluminescence kit ECL (Amersham, UK) for 1-5 minutes. X-OMAT AR film(Kodak Co., Rochester, N.Y.) is used to detect the chemiluminescence.

PCR Amplification of Rearranged bcl-2

Nested oligonucleotide amplification is performed at the MBR or mcr ofthe bcl-2/Ig_(H) hybrid gene using previously published methods (23).Briefly, samples containing 1 μg of genomic DNA are initially amplifiedfor 25 cycles in a final volume of 50 μg containing 50 mmol/L KCl, 10mmol/L Tris HCL, 2.25 mmol/L MgCl₂, 200 mmol/L oligonucleotide primers,200 mmol/L each of dGTP, dCTP, dTTP and dATP, and 1.5 U Taqpolymerase(Cetus, Emeryville, Calif.). Reamplification of an aliquot ofproduct is performed for 30 cycles in a final volume of 50 μl usingidentical conditions to the original amplification, with oligonucleotideprimers internal to the original primers. Aliquots of the final productare analyzed by gel electrophoresis in 4% agarose gels containingethidium bromide and visualized under UV light. DNA is Southern blottedonto Zeta-probe blotting membrane (BioRad. Richmond, Calif.) andbcl-2-specific DNA is detected by hybridization with oligonucleotideprobes radiolabeled with ³²P(ATP) using T4 polynucleotide kinase.

Removal of Patients from Protocol Therapy

Patients will be removed from protocol for any of the following reasons:

Unacceptable toxicity (as defined above).

The patient declines further therapy.

The patient experiences progressive lymphoma.

It is deemed in the best interest of the patient. In this instance,

-   -   The Principal Investigator should be notified.    -   The reasons for withdrawal should be noted in the flow sheet.        Response Criteria

Patients will be reevaluated for tumor response after every two cyclesof chemotherapy using the following criteria:

Complete Response—disappearance of all clinical and laboratory(excluding PCR) signs and symptoms of active disease for a minimum ofone month.

Partial Response—a 50% or greater reduction in the size of the lesionsas defined by the sum of the products of the longest perpendiculardiameters of all measured lesions lasting for a minimum of one month. Nolesions may increase in size and no new lesions may appear.

Minimal Residual Response—a ≧90% partial response. For most patients inthis category, this will mean≦10% residual bone marrow involvement bylymphoma.

Progressive Disease—an increase of 25% or more in the sum of theproducts of the longest perpendicular diameters of all measuredindicator lesions compared to the smallest previous measurement or theappearance of a new lesion.

Drug Formulation and Toxicity Data

Cyclophosphamide (CTX. Cytoxan)-NSC #26271

-   -   Source and Pharmacology—CTX is an alkylating agent, related to        nitrogen mustard, which is biochemically inert until it is        metabolized to its active components by the liver        phosphoramidases. It is non-phase-specific. The drug is excreted        exclusively by the kidney after parenteral administration.    -   Formulation and Stability—CTX is supplied as a 100, 200, 500,        1000 mg and a 2 gram lyophilized powder with 75 mg mannitol per        1 00 mg (anhydrous) cyclophosphamide. The vials are stored at        room temperature (59-86° F.) and reconstituted with sterile        water for injection to yield a final concentration of 20 mg/l nl        as described in the package insert. Reconstituted        cyclophosphamide is stable for at least 6 days under        refrigeration and for 24 hours at room temperature.        Reconstituted drug and diluted solutions should be stored under        refrigeration.    -   Supplier—Commercially available.    -   Route of Administration—The cyclophosphamide used in this        regimen is given IV over 30 minutes and is diluted in 100 cc of        either D₅W or NSS.    -   Toxicity—Toxicities described with cyclophosphamide include        nausea, vomiting, myelosuppression, gonadal failure in both        males and females, alopecia, interstitial pneumonitis, pulmonary        fibrosis, hemorrhagic cystitis, cardiac events (cardiomyopathy),        syndrome of inappropriate antidiuretic hormone secretion (SIADH)        and rarely, anaphylaxis.

Prednisone (Deltasone. Meticorten, Liquid Pred) NSC#10023

-   -   Source and Pharmacology—Prednisone is the synthetic congener of        hydrocortisone, the natural adrenal hormone. It binds with        steroid receptors on the nuclear membrane, blocks mitosis, and        inhibits protein synthesis. It kills primarily during the        S-phase of the cell cycle. It is catabolized in the liver and        excreted in the urine. Peak blood levels occur within two hours        after oral intake. Plasma half-life is 3-6 hours. (Biologic        half-life is 12-30 hours.)

Cortisone 25 Equivalent Hydrocortisone 20 strength in mg Prednisone 5Decadron 0.75

-   -   Formulation and Stability—Available in 1, 2.5, 5, 10, 20 and 50        mg tablets; 5 mg/5 ml liquid.    -   Supplier—Prednisone is commercially available.    -   Route of Administration —PO; NOTE: May cause GI upset; take with        meals or snacks. Take in the morning prior to 9 a.m.    -   Toxicity—Toxicities described with prednisone include fluid and        electrolyte changes, edema, hypertension, hyperglycemia,        gastritis, osteoporosis, myopathy, behavioral and mood changes,        poor wound healing, and Cushing's syndrome (moon face, buffalo        hump, central obesity, acne, hirsutism and striae).

VP-16 (Etoposide. VePesid) NSC#141540

-   -   Source and Pharmacology—VP-16 is a semisynthetic derivative of        podophyllotoxin which inhibits topoisomerase II and functions as        mitotic inhibitor, but does not bind microtubules. Its main        effect appears to be in the S and G₂-phase of the cell cycle.        The mean terminal half-life is 11.5 hours, with a range of 3 to        15 hours. It is primarily excreted in the urine.    -   Formulation and Stability—VP-16 is supplied in vials containing        either 100 or 500 mg of etoposide (20 mg/ml) in a polyethylene        vehicle. VP-16 is diluted in either 500 cc of 5% dextrose or        0.9% Sodium Chloride Injection. Diluted solutions        (concentrations of 0.2, 0.4 mg/ml and 1 mg/ml) are stable for        96, 48 hours and 2 hours, respectively at room temperature under        normal room fluorescent light in both glass and plastic        containers. Do not refrigerate etoposide-containing solutions.    -   Supplier—VP-16 is commercially available.    -   Route of Administration—Etoposide is administered as an IV        infusion over 60 minutes.    -   Toxicity—Toxicities described with etoposide administration        include myelosuppression (neutropenia), nausea, vomiting,        mucositis, allergic reactions characterized by anaphylactic        symptoms and hypotension and alopecia.

Doxorubricin (Adriamycin) NSC #123127

-   -   Source and Pharmacology—Doxorubicin is an anthracycline        antibiotic isolated from cultures of Streptomyces peucetius. It        binds to DNA and inhibits nucleic acid synthesis, with its major        lethal effect occurring during the S-phase of the cell cycle.        Since it is primarily excreted by the liver, any liver        impairment may enhance toxicity. Some of the drug has a very        short α T ½ of <20 minutes and a β ½ of 17 hours. Animal studies        indicate cytotoxic levels persist in tissue for as long as 24        hours. Biliary excretion also is a source of elimination for        Doxorubicin; therefore, patients with        hyperbilirubinemia/cholestasis caused by something other than        lymphoma should have dosage modification.    -   Formulation and stability—Doxorubicin is available as a        freeze-dried powder in 10, 50 and 150 mg vials. The drug is        stored at room temperature, protected from light, and is        reconstituted with sodium chloride 0.9% (NSS) to yield a final        concentration of 5 mg/ml. The reconstituted solution is stable        for 7 days at room temperature (15-30° C.) or if stored under        refrigeration (2-8° C.).    -   Supplier—Doxorubicin is commercially available.    -   Route of Administration—Doxorubicin is given as a slow IV        injection over 5-7 minutes through an established line with a        free flowing IV.    -   Special precautions: Avoid extravasation and local contact with        skin or conjunctiva.    -   Toxicity—Toxicities described with doxorubicin administration        include myelosuppression, nausea, vomiting, mucositis,        stomatitis, alopecia, diarrhea, facial flushing, dose-related        congestive cardiomopathy, arrhythmias, vein streaking        (hypersensitivity reaction), radiation-recall dermatitis, local        cellulitis, vesication and tissue necrosis upon extravasation        (SQ and dermal necrosis).

ID-KLH Vaccine

-   -   Source—Idiotype protein from the individual B cell lymphomas is        obtained from tissue culture, purified, and covalently coupled        to keyhole limpet hemocyanin (KLH) as previously described. Each        batch is produced according to Good Manufacturing Practices        standards and tested for sterility, endotoxin contamination, and        general safety prior to its use in any patient. The preparation        and quality control/quality assurance testing of the Id-KLH        conjugate is performed by TSI Washington under CRB contract. The        IND for the Id-KLH vaccine will be held by the Drug Regulatory        Affairs Section, CTEP.    -   How supplied—Formulated product for subcutaneous administration        contains 0.5 mg of Id and KLH each per ml of normal saline.        Id-KLH is supplied as a 1 ml vial.    -   Storage—Prior to administration, Id-KLH is stored at −20° C.    -   Administration—After thawing and gentle agitation, the vial        contents are drawn up using an 18-gauge needle on a syringe.        After the entire contents have been drawn up, the 18-gauge        needle is replaced by a 25-gauge needle for injection. This        procedure is important to ensure that all particulates (normal        components of this vaccine) are obtained from the vial.    -   Toxicity—Toxicities described with Id-KLH vaccine administration        include local site reactions (erythema, induration, swelling and        tenderness), fever, chills, rash, myalgias and arthralgias. Mild        elevations in creatinine phosphokinase (CPK) have been observed.

GM-CSF (Sargramostim: NSC #613795; BB-IND 2632

-   -   Source and Pharmacology—The GM-CSF used in this study is        glycosylated, recombinant human GM-CSF. This GM-CSF is an        altered form of the native molecule; the position 23 arginine        has been replaced with a leucine to facilitate expression of the        protein in yeast (Saccharomyces cerevisiae).    -   Formulation and Stability—The GM-CSF is formulated as a white        lyophilized cake and is provided in vials containing 500 μg of        the GM-CSF protein as well as 10.0 mg of sucrose, 40.0 mg of        mannitol, and 1.2 mg of Tris (Trimethamine).    -   To prepare a vial of GM-CSF for direct subcutaneous use,        aseptically inject 1.0 ml of Sterile Water for Injection, USP,        into the vial to dissolve the lyophilized cake. The diluent        should be directed against the side of the vial to avoid excess        foaming. Avoid vigorous agitation of the vial; do not shake.        This yields a solution containing 500 μg/1 ml. The        unreconstituted material should be kept refrigerated at 2-8° C.        and is stable for at least eighteen months. Once reconstituted,        the solution is stable for at least 24 hours at 2-8° C. or at        18-25° C. Because the product does not contain a preservative,        vials should be treated as unit-dose containers; reconstituted        solution should be held at 2-8° C. and discarded after no more        than six hours. Do not freeze GM-CSF.

Supplier. Manufactured by Immunex.

-   -   Route of Administration—The appropriate total dose is withdrawn        into and administered from a plastic tuberculin syringe. The        GM-CSF is injected subcutaneously as close as possible to the        Id-KLH injection site. All GM-CSF doses for each patient are        administered by the nursing staff in the outpatient unit.    -   Toxicity—Toxicities described in patients receiving GM-CSF        include: fever, chills, diaphoresis, myalgias, fatigue, malaise,        headache, dizziness, dyspnea, bronchospasm, pleural effusion,        anorexia, indigestion, nausea, vomiting, diarrhea, injection        site tenderness, urticaria, rash, pruritus, hypersensitivity        reaction, bone pain, thromboembolic events, phlebitis,        hypotension, peripheral edema, leukocytosis, thrombocytosis or        thrombocytopenia, hepatic enzyme abnormalities, and bilirubin        elevation. The first administration of GM-CSF has provoked a        syndrome of dyspnea and hypotension within two hours after        GM-CSF injection in a single patient receiving yeast-derived        GM-CSF; this type of reaction has more frequently been observed        in patients receiving GM-CSF produced in E. coli. One report of        a vascular leak-like syndrome occurring after autologous bone        marrow transplant in a patient receiving continuous IV infusion        of GM-CSF has been recorded.

Unconjugated Lymphoma Immunoglobulin Idiotype (for intradermal skintesting) NSC# 684151

-   -   Source—The patient-specific purified idiotype protein,        previously produced according to GMP standards as described        above, is vialed as a separate product by TSI Washington        Laboratories and will be supplied by CTEP, DCT, NCI. This vialed        product is tested separately for sterility, endotoxin, and        mycoplasma, according to IND specifications previously discussed        with the FDA.    -   Each vial of patient-specific unconjugated idiotype will be        labeled to include the following information:        -   Purified sterile immunoglobulin idiotype            -   patient-specific lot        -   final volume and concentration of product        -   patient-specific immunoglobulin subtype        -   storage conditions        -   fill date        -   patient identification (first name/last initial)    -   How Supplied—This product is available as a solution containing        0.2-0.3 ml of unconjugated idiotype diluted in sodium chloride        0.9%. The solution is contained inside a sterile vial. The final        solution contains 0.5 mg of patient-specific immunoglobulin        idiotype protein. Intact vials are stored at −20° C.    -   Toxicity—The toxicities associated with administration of        unconjugated Id protein are anticipated to be identical to those        described with the Id-KLH vaccine.        -   The safety issues regarding the injection of heterologous            idiotype protein isolated from other patients' B-cell tumors            have already been fully addressed in CRB # 9407 (NCI            T94-0085; Active immunization of Healthy Sibling Marrow            Transplant Donors With Myeloma-derived Idiotype) and are            felt to be minimal, because of the highly purified nature of            the protein.        -   Briefly, an immune response of any consequence to the            isotype matched idiotype used as a negative control during            the second skin test is not likely, based on:        -   1. The isotype matched idiotype will only be administered            once and is not conjugated to a carrier protein. These            minimize the chance of eliciting a sustained immune response            to the protein.        -   2. Any immune response specifically directed against the            idiotype (i.e., variable region) on the control idiotype            protein is not likely to cross-react with host cells and is            therefore not likely to be of any consequence.        -   3. An autoimmune response against constant region or            allotype determinants shared between the idiotype of the            patient's own tumor and that of the control idiotype tumor            is theoretically possible. However no evidence of such            autoimmune responses have been observed either in vivo or in            vitro during the course of immunization of sibling bone            marrow transplant donors with purified myeloma protein.        -   Furthermore, a safety precedent exists for immunizing            patients with material derived from tumor cells from other            patients. For example, in attempting to develop immune            responses against metastatic melanomas, patients were            immunized with 1) intact melanoma cells; 2) shed antigens            fractionated by detergent treatment and            ultracentrifugation; 3) melanoma cells infected with            vaccinia virus and melanoma cells freeze thawed and            mechanically disrupted, all using a pool of allogeneic            melanoma cell lines (24-28).

Bactrim will be supplied by the Clinical Center.

Filgrastim (G-CSF)/Neupogen

-   -   Source and Pharmacology—The G-CSF to be used in this study is        the recombinant methionyl human granulocyte-colony stimulating        factor (r-methi-HuG-CSF). G-CSF is a hematopoietic growth factor        with effects on both immature bone marrow progenitors and mature        myeloid cells. It acts by supporting growth of human bone marrow        derived colony forming units and enhancing neutrophil growth and        proliferation.    -   Formulation and Stability—The G-CSF is formulated as a clear,        sterile solution and is provided in vials at a final        concentration of 300 mcg/ml. The commercial vials are available        in 300 and 480 mcg sizes. The intact vials are stored under        refrigeration (2-8° C.) prior to use and must not be frozen and        are stable at this temperature for at least one year.    -   Supplier—Manufactured by Amgen; supplied by the Clinical Center.    -   Route of Administration—The appropriate total dose is withdrawn        into and administered from a plastic tuberculin syringe. The        G-CSF is injected as a subcutaneous injection. The patient or        other care-giver is instructed on proper injection technique.    -   Toxicities—Toxicities described with G-CSF include: transient        bone pain (sternal/pelvic) myalgias, fatigue, mild elevations in        uric acid, LDH and alkaline phosphate, fluid retention,        transient hypotension, local inflammation at injection site,        rarely cutaneous vasculitis, rarely pericardial effusion and        rare anaphylactic reactions with first dose.        Statistical Considerations

Statistical issues to be addressed include identification of significantendpoints, sample size determination, power considerations,stratification, randomization and design.

The design of this study is viewed primarily within the framework of aSingle Arm Phase II trial. However, as the purpose is also toinvestigate possible differences between GM-CSF doses as adjuvants, itincorporates design elements characteristic of a Multiple Arm Phase IIor a randomized Phase III trial. Statistical methods that areappropriate to both single and double arm designs are described.

Patients receive combination chemotherapy to best response followed byId-KLH combined with GM-CSF. Several outcome measures (endpoints) areevaluated in order to meet the objectives of this study. They include:

-   -   1) The clinical complete response rate (in contradistinction to        the molecular or PCR response rate) of all patients to ProMACE—a        percentage indicated by the disappearance of all clinical and        laboratory signs and symptoms of active disease, excluding PCR,        for a minimum of one month.    -   2) The Polymerase Chain Reaction (PCR) response rate        (molecular-complete response rate)−the percentage of patients        who, having achieved a clinical complete response still remain        PCR (+) at the end of chemotherapy, and who then become PCR (−)        with the administration of immunotherapy.    -   3) Disease Free Survival Rate—computed by Kaplan-Meier curves        and related survival measures.

The PCR response rate is taken as the primary outcome variable ofinterest to ascertain the following: (1) to determine the ability of Idimmunization to eradicate bcl-2 positive tumor cells from the bonemarrow and; (2) to identify the more biologically active of the twodoses of GM-CSF. In this endeavor, the plan is to accrue 42 patients. Itis estimated that approximately 38 (90%) of these patients will be bcl-2(+) and thus evaluable for molecular response rate. The other fourpatients may still be evaluable for a molecular response rate based onIg gene amplification using allele-specific (CDR3) primers by PCR. Fromprevious experience with ProMACE-based regimens, it is estimated that 32(85%) of these patients will achieve either a complete response(complete clinical response, CCR) or a partial response in which a >90%partial remission has been obtained (high partial response, HPR). Theaccuracy of these estimates are of some interest. For the 42 (90%)patients anticipated to be bcl-2 (+), lower and upper 95% confidenceintervals are 77% and 96%. For the 38 (85%) patients anticipated toachieve either a complete clinical response or a high partial response,the lower and upper confidence intervals are 70% and 93%.

Patients are stratified on the basis of their ProMACE treatmentperformance as either a complete clinical responder (CCR) or as a highpartial responder (HPR). It is not known exactly what percentage ofthese 32 patients will be CCRs and what percentage will be HPR'S. Hencea block size of four (4) is used in the randomization scheme to assure areasonably balanced allocation to each dose group. Given the patientsallocation stratum, he (she) is randomly assigned to one of the adjuvantgroups according to the envelope method (29). Specifically, a block offour assignments is placed in four separate envelopes. The block of fouris placed in one of the two allocation strata, say CCR. Another block offour is placed in the other allocation strata, say CCR. Another block offour is placed in the other allocation stratum, HPR. When a patient isto be randomized, a call is made to the biostatistician who, after beinginformed of the patients status as either a CCR or an HPR, randomlydraws an envelope from the appropriate stratum to determine the patientsdose group assignment. After the four envelopes pertinent to aparticular stratum have been exhausted, the next batch of four envelopesis made available for use. This procedure is continued until a total of32 patients have been assigned to the two dose groups.

For example, it is estimated that 50-80 percent of pathological completeresponders will fall into the CCR category. If 75% of 32, or 24 patientswere to be classified as CCRs, six blocks of four envelopes would berequired to randomly assign 12 patients to cohort 1 and 12 patients tocohort 2. A similar procedure would occur concurrently with the 8patients classified as HPRs. Two blocks of four envelopes would berequired to randomly assign 4 patients to cohort 1 and 4 patients tocohort 2. At no time could the number of patients in each dose groupdiffer by more than four.

At the time of data analysis, approximately 16 subjects will compriseeach dose group and a test for the difference in PCR response ratesbetween the two groups will be conducted. By hypothesis, neither dosegroup is predicted to have a higher PCR response rate than the other;hence, a two-tailed test is appropriate. Power calculations show that,with the groups limited to 16 patients, the difference in PCR responserates will have to be large (30, 31). For example, to detect adifference at the α=0.05 level of significance with power (1-β) equal to80%, the response rates must differ by 55%; with power equal to 50%, theresponse rates must differ by 50%. In the event that no significantdifference is detected, the subjects will be pooled and the overall PCRresponse rate will be assessed. With a total of 32 CCRs and HPRs treatedwith vaccine, the width of a two-tailed 95% confidence interval for aresponse rate of 50% will not exceed 17 percentage points. If the actualresponse rate is higher or lower than 50%, the confidence interval willbe smaller.

Disease-free survival distributions are estimated by the Kaplan-Meier(product-limit) method and dose groups are compared using the log ranktest. If no dose group differences are detected, the subjects from bothgroups are pooled and the Kaplan-Meier estimate of the survivorshipfunction and related functions are evaluated. If suggested by the dataanalysis, parametric distributions (e.g., Weibull, log-normal) are fitas well (32, 33).

Research ethics: Subjects from [both genders and] all racial/ethnicgroups are eligible for this study if they meet the eligibility criteriaoutlined above. To date, there is not information that suggests thatdifferences in grud metabolism or disease response would be expected inone group compared to another. Efforts are made to extend accrual to arepresentative population, but in this preliminary study, a balance mustbe struck between patient safety considerations and limitations on thenumber of individuals exposed to potentially toxic and/or ineffectivetreatments on the one hand and the need to explore gender and ethnicaspects of clinical research on the other hand. If differences inoutcome that correlate to gender or to ethnic identity are noted,accrual can be expanded or a follow-up study can be written toinvestigate those differences more fully. Alternatively, substantialscientific data exist demonstrating that there is no significantdifference in outcome between genders or various ethnic groups.

Records to be Kept and Quality Assurance

Consent form: The original signed informed consent documents will bekept with the patient's other study documentation (e.g., the researchchart). A copy of the informed consent document will also be retained bythe Data Management Section.

The Clinical Coordinator, Data Management Section, will ascertain thedates of the IRB approvals before registering the first patient.

It is understood that the disclosed invention is not limited to theparticular methodology, protocols, and reagents described as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells, reference to “theantibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are as described. Publications cited herein andthe material for which they are cited are specifically incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

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1. A fusion polypeptide comprising a defensin and a human tumor antigen.2. The polypeptide of claim 1, wherein the human tumor antigen is ahuman B cell lymphoma tumor antigen.
 3. The polypeptide of claim 2,wherein the defensin is an alpha defensin or a beta defensin.
 4. Thepolypeptide of claim 3, wherein the defensin is a beta defensin selectedfrom the group consisting of HBD 1 and HBD2, or an alpha defensinselected from the group consisting of HNP-1, HNP-2, and HNP-3.
 5. Thepolypeptide of claim 1 wherein the human tumor antigen is human Muc-1.6. The polypeptide of claim 1, wherein the defensin is an alpha defensinor a beta defensin.
 7. The polypeptide of claim 6, wherein the defensinis a beta defensin selected from the group consisting of HBD 1 and HBD2,or an alpha defensin selected from the group consisting of HNP-1, HNP-2,and HNP-3.
 8. The fusion polypeptide of claim 1, wherein the defensin isa beta defensin, and wherein the tumor antigen is a B cell lymphomatumor antigen.
 9. The fusion polypeptide of claim 8, wherein the betadefensin is human beta defensin 2 (HBD2).
 10. The fusion polypeptide ofclaim 2, wherein the B cell lymphoma tumor antigen is selected from thegroup consisting of an antibody, a single chain antibody and an epitopeof an idiotype of an antibody.
 11. The fusion polypeptide of claim 10,wherein the B cell lymphoma tumor antigen is sFv38.
 12. A fusionpolypeptide comprising a defensin and the amino acid sequence of SEQ IDNO:
 1. 13. A composition comprising the fusion polypeptide of claim 1 ina pharmaceutically acceptable carrier.
 14. A composition comprising thefusion polypeptide of claim 3 and a pharmaceutically acceptable carrier.15. An isolated nucleic acid encoding the fusion polypeptide of claim 1.16. A vector comprising the nucleic acid of claim
 15. 17. An isolatedhost cell comprising the vector of claim
 16. 18. An isolated nucleicacid encoding the fusion polypeptide of claim
 3. 19. A vector comprisingthe nucleic acid of claim
 18. 20. An isolated host cell comprising thevector of claim
 19. 21. An isolated nucleic acid encoding the fusionpolypeptide of claim
 10. 22. A vector comprising the nucleic acid ofclaim
 21. 23. An isolated host cell comprising the vector of claim 22.24. An isolated nucleic acid encoding the fusion polypeptide of claim12.
 25. A vector comprising the nucleic acid of claim
 24. 26. Anisolated host cell comprising the vector of claim
 25. 27. A compositioncomprising the nucleic acid of claim 15 and a pharmaceuticallyacceptable carrier.
 28. A composition comprising nucleic acid of claim18 and a pharmaceutically acceptable carrier.
 29. A method of producingan immune response in a subject, comprising administering to the subjectan effective amount of the composition of claim 13, thereby producingthe immune response in the subject.
 30. The method of claim 29, whereinthe immune response is an effector T cell (cellular) immune response.31. A method of producing an immune response in a subject, comprisingadministering to the subject an effective amount of the composition ofclaim 27, under conditions whereby the nucleic acid of the compositioncan be expressed, and thereby producing the immune response in thesubject.
 32. The method of claim 31, wherein the immune response is aneffector T cell immune response.
 33. A method of producing an immuneresponse in a subject, comprising administering to the subject aneffective amount of a composition comprising the fusion protein of claim8, thereby producing the immune response in the subject.
 34. The methodof claim 33, wherein the beta defensin is human beta defensin 2 (HBD2).